Science against microbial pathogens: communicating current research and technological advances
______________________________________________________________________________
A. Méndez-Vilas (Ed.)
Immobilized Antimicrobial Agents: A Critical Perspective
John-Bruce D. Green,1 Timothy Fulghum and Mark A. Nordhaus
1
Technology Resources, Baxter Healthcare Corporation, Round Lake, Illinois 60073, USA
Traditionally antimicrobial devices and applications have leveraged the elution of biocides from devices which were
infused with these active molecules. The elution of these agents can, in some cases, limit the application of the device and
can potentially lead to unwanted side effects. Following the ground breaking works of Isqueth, Abbott and Walters,
surface immobilized antimicrobial agents (iAMA) have been explored. Devices sporting immobilized agents may remain
active longer and minimize side effects. Researchers have examined the structure-activity of antimicrobial agents
appropriate for immobilized applications. A great deal of effort goes into the design, synthesis, immobilization and
characterization of the AMA’s, and yet it is common for the activity measurement to be off-the-shelf methods that are
poorly aligned with the needs of immobilized agents. As a result, much time and effort is wasted developing structureactivity relationships based on questionable interpretations. The purpose of this review is to educate and guide researchers
planning to develop immobilized antimicrobial agents. As such, the focus is not only to review the agents which have
been immobilized, but also to highlight the key means of immobilization and importantly the activity testing methods.
Keywords immobilized antimicrobial agent; efficacy testing, immobilization strategies,
Introduction
Microbial contamination and infection pose a serious concern for food, military and medical industries. The impact to
healthcare is undisputed, with hospital associated infections (HAIs) from bacterial, fungal and viral agents contributing
to ~100,000 deaths annually in the USA alone[1,2]. The first line of defense against food spoilage and medical
infections is the avoidance of microbial load, followed by the appropriate conditions that minimize microbial growth.
Additional protection can be added through the use of containers and devices that possess antimicrobial or antifouling
properties.
Most of the antimicrobial applications have exploited eluting agents[3-7] such as benzalkonium chlorides,
cetylpyridinium chloride, aldehydes, anilides, diamidines, silver, chlorhexidine, triclosan, N-halamines, and povidoneiodine. While such agents are known to be efficacious and appropriate for specific applications, their extension to some
medical device applications may be hindered by the elution of the agent, due to a limited reservoir capacity or potential
side-effects caused by these extractables. Immobilized antimicrobial agents offer an alternative motif which eliminates
patient exposure to elutable, active agents, and potentially increases the duration of antimicrobial efficacy[8,9]. A wide
range of antimicrobial agents have been immobilized including small molecules (e.g., quaternary ammonium
silanes)[10-17], quaternary ammonium polymers[18-35], polyamines[36-42], chitosan[43-49], enzymes[50-55],
peptides and peptide mimetics[56-65]. These agents have been immobilized on a host of surfaces including metals,
plastics, as well as natural and man-made fabrics[66].
This chapter will guide the reader through the various antimicrobial agents that have been immobilized, and then
touch on some key points such as mode of action and the testing methods. These last points are especially relevant for
researchers who are developing new materials or are evaluating established materials.
The antimicrobial mode of
action[2,67-75] for an agent is expected to be affected by the process of immobilization, since the chemical composition
and dimensions of the extra-cytoplasmic bacterial components (membranes, peptidoglycan wall, capsule, fimbriae and
flagella if present), are relevant to the performance of surface tethered AMAs. For example, given that the mode of
action for tetracycline involves disruption of the binding between 16S rRNA and tRNA[76], surface immobilization via
a short tether severely restrict it’s access from the cell interior, thus dramatically reducing if not eliminating the AMA’s
efficacy. Once immobilized, the appropriate choice of test method and the interpretation of the results can be
complicated by numerous variables. This chapter will attempt to provide insight into these factors and assist researchers
in the appropriate choice of test methods for their immobilized AMA samples.
Immobilization Strategies
There are a number of different strategies for immobilization of AMA to substrates: (1) “graft-to” strategies involve the
covalent coupling of the intact AMA to a surface; (2) “physical adsorption” methods involve physisorption of the AMA
through non-covalent but strong or multidentate interactions at the surface; (3) “surface initiated” strategies involve the
synthesis of the AMA from initiators covalently immobilized to the surface; and (4) “as-formed” methods involve
creating a substrate which contains the AMA at the time the substrate is formed. Frequently researchers employ a
multi-step process that involves two or more strategies, for example graft-from synthesis of polymer brushes which are
then reacted with AMA’s via graft-to pathways. Graft-to strategies begin with the synthesis or purchase of a potentially
84
©FORMATEX 2011
Science against microbial pathogens: communicating current research and technological advances
_______________________________________________________________________________
A. Méndez-Vilas (Ed.)
surface reactive AMA. Frequently, the surface requires an activation process that generates amine, carboxylic acid,
aldehyde or thiol functionalities. The AMA can be coupled to the activated substrate by use of heterobifunctional
linkers such as succinimide, carbodiimide, maleimide, or aldehyde[77]; though sometimes the linking uses clickchemistry[78]. Sometimes these linkers contain polymer spacers such as polyethyleneglycol, which serve to enhance
the degrees of freedom for the AMA, thereby enabling more modes of action and increasing the efficacy. While the
graft-to methods appear strong and irreversible by the nature of the covalent bonds, sometimes these bonds can be
hydrolyzed or broken by mechanical forces. Physisorption can sometimes be stronger and more irreversible than
covalent bonding because of multi-dentate interactions such as hydrogen bonding, ionic bonding, or even steric
interactions caused by entanglement during solvent swelling of polymer films. The best examples of robust physisorbed
films are the layer-by-layer or LbL films[79], which are often combined with eluting strategies, but some cases use
quaternary ammonium or other AMA as the outer layer of the LbL system[80]. Other physical absorption methods
involve exploiting the charge pairing or strong ionic bonding to hold smaller AMA to a substrate with opposite charge.
When the physical interaction involves ionizable groups, the pH of the environment is of key importance to the stability
of the film. In as-formed strategies the molecules are incorporated during the substrate formation process, and become
crosslinked or entangled within the substrate polymers. Nagel and coworkers demonstrate that surface reactive
injection molding can generate permanently modified parts, in this case polycarbonate with PEI presented at the
surface[81]. Namba et al., included an AMA within the ingredients for methacrylic polymerization, thus entangling the
AMA within the substrate matrix[12]. Unlike the above methods, surface initiated or graft-from strategies provide a
higher degree of control over the immobilization process. In these methods, molecules are synthesized directly from the
surface, as in the case of solid phase synthesis of peptides or oligonucleotides. Well defined polymeric structures have
been created through “living” or controlled polymerization techniques[82], such as reversible addition fragmentation
chain transfer polymerization (RAFT) [83,84], nitroxide mediated polymerization (NMP)[85] and metal catalyzed
living radical polymerizations like atom transfer radical polymerization (ATRP)[86]. In a similar but substantially more
controlled manner, researchers can use solid phase synthesis methods to graft specific sequences of peptides and
peptoids directly from a substrate. Cellulose-amino-hydroxypropyl ether (CAPE) has been used to synthesize
antimicrobial peptides (AMP) directly on cellulose substrates. Sometimes researchers use multi-step methods by
combining strategies. In one example, researchers combined graft-to and surface-initiated methods to modify substrates
with complex polymer films[38].
Figure 1 Some immobilization schemes. (A) Physical
adsorption methods by noncovalent perhaps multidentate
interactions e.g., LbL films, block polymers, etc. (B) Graft-to
methods by creating covalent bonds with the surface e.g.,
PEIs, AMPs, enzymes. (C) Graft-from or surface-initiated via
synthesis of AMA from initiator at surface perhaps by ATRP
e.g., PVP, PDMAEMA, methacrylates. From Barbey et al.
Chemical Reviews. 2009;109(11):5437-5527.
Immobilized Agents
A wide range of molecules have been immobilized and tested as antimicrobial agents including amine containing
polymers, quaternary ammonium polymers, guanides, enzymes, chitosan, peptides, peptoids and other peptide
mimetics. The following sections highlight these different classes of agents. The literature is full of potential
explanations for the mechanism or mode of action for each of these agents, and much of these hypotheses are based
upon the results from standard efficacy tests. These AMA specific sections will briefly touch on the test methods, but
they will be covered in more depth and with more critical assessment later. The field of immobilized AMAs has become
quite expansive. This review is not able to cite everything, but instead aims to highlight those key publications.
©FORMATEX 2011
85
Science against microbial pathogens: communicating current research and technological advances
______________________________________________________________________________
A. Méndez-Vilas (Ed.)
Quaternary ammonium silanes and other small molecules
Perhaps the most well-known immobilized antimicrobial agent is the silane (3-trimethoxysilyl)
propyldimethyloctadecyl ammonium chloride[10], Si-QAC. This molecule, as with any tri-alkoxy silane, can
autopolymerize to form long branched polymer chains with an (-Si-O-) backbone. The quaternary ammonium side
chains on this siloxane polymer form a similar motif to the quaternary ammonium polymers discussed below. In the
initial work on this molecule, the silane was bound to a wide range of different substrates, including siliceous surfaces,
man-made fibers, natural fibers, metals and assorted industrial materials[11]. These modified surfaces were tested
against a host of microbes; including bacteria (both gram positive and negative), yeast, algae and fungi. A modified
ASTM 2149E and an aerosol method were used to probe antimicrobial efficacy, and radiolabeling was used to prove
that the agent did not elute from the surface. This initial work was followed by more detailed publications of the silane
immobilized to different substrates and under different application conditions[14]. Each treatment was slightly different,
and the resulting films may be different from the films created by the initial researchers. In one example of note, SiQAC was reacted silicone rubber, and using a flow cell combined with rinsing and live-dead staining, they clearly
demonstrated that the modified surfaces supported more dead bacteria than the unmodified controls[16]. Whether this
was due to a causal based surface killing of the bacteria, or an enhanced adhesion of membrane compromised cells to
the surface is unclear. The effect was even observed after the surfaces were exposed to human plasma. In even more
extreme applications, the authors performed a series of in vivo experiments, and they observed that the samples
inoculated ex vivo were efficacious while those inoculated in vivo were not.
In addition to these quaternary ammonium silanes, other small molecules such as aminoglycoside antibiotics and
cetylpyridinium chloride (CPC) have been immobilized[12],[13]. However, in each case whether immobilized by
covalent coupling or incorporated in as-formed films, these examples usually show tell-tale signs of elution of the
agents. As such, even though some of the agents may have been immobilized and may remain associated with the part
throughout the experiment, these surfaces are likely efficacious via the elution of AMAs. In some cases this was
evidenced by ZOI data, and in some by the basic absence of any physical mechanism to immobilize the small soluble
efficacious molecules.
Quaternary ammonium polymers
The most thoroughly studied class of AMAs are the quaternary ammonium polymers[18-21], PQAs, with polymer
backbones such as polyethyleneimine (PEI), polyvinylpyridinium (PVP), chitosan, and assorted acrylates. These
polymers have been immobilized by virtually every method listed above, and are often modified by post immobilization
reactions. Much of the graft-to work has involved PVP and PEI using the immobilization process to control the
polymer surface density and subsequent on-surface quaternization with different sidechains and
counterions[17,22,30,31]. Other groups have focused efforts on surface initiated polymerization using controlled
ATRP reactions[24,26-29]. The works from the Klibanov and Russell groups have systematically probed the
antimicrobial impact of key properties such as surface charge density, polymer chain length, polymer chain density,
counter ion identity, and quaternary ammonium sidechain length.
Aliphatic quaternary ammonium polymers
The most common aliphatic polymer systems studied have been polyethyleneimine (PEI) and its derivatives. It is
common to immobilize the PEI by graft-to methods, although some groups have used as-formed methods akin to
painting to create a thick film of the PEI. The painting process did not immobilize the molecules covalently, but instead
they exploited the inherently poor solubility of these polymers as a barrier to dissolution in the inoculum. Two specific
studies focused on the length of the sidechain and the charge of functional groups[23],[35]. The PEI immobilization
was followed by alkylation, acylation or carboxyalkylation to generate cationic (quaternary ammonium), neutral
(amide), and zwitter-ionic (quaternary ammonium carboxylic acid) functional groups respectively. Alkylation was
performed with a range of chainlengths (ethyl, butyl, hexyl, dodecyl and octadecyl) followed by subsequent methylation
to quaternize the amine. Their results showed that positive charge was necessary, and that neither the neutral nor the
zwitter-ionic surfaces were efficacious. They also concluded that when terminally methylated, the chainlength of the
alkylating group should be greater than n-butyl for a high efficacy. The covalent strategy found similar results for both
Gram positive (S. aureus and S. epidermidis) and negative (E. coli and P. aeruginosa) bacteria. The painted study
demonstrated efficacy of the cationic polymers against aerosol based microbes (E. coli, S. aureus, and influenza virus)
with a greater than 4 log reduction in CFU or PFU within minutes of exposure. In an effort to demonstrate that leaching
was not impactful, the authors performed two tests (1) an extraction from a painted sample, and (2) an extraction from
200mg/mL of the pure agents. In each case the bulk “extraction” buffer solution was then inoculated with microbes,
and tested for efficacy. As with many elution tests, this probes the amount of material that can dissolve into the
aqueous phase and asks whether this bulk solution concentration is adequate to impact the efficacy test. The second
method is much stronger as it is essentially a saturated solution of the agent, while the first method may still have
locally high concentration at the surface due to the diffusion gradient slowly increasing the bulk solution concentration.
86
©FORMATEX 2011
Science against microbial pathogens: communicating current research and technological advances
_______________________________________________________________________________
A. Méndez-Vilas (Ed.)
However both methods are expected to have concentrations much less than the apparent concentration at the interface.
Interestingly, one group examined the bacterial resistance (or lack thereof) with respect to these quaternized PEI
surfaces[30]. Surfaces with PEI that was hexyl,methyl quaternized, showed efficacy against both E. coli and S. aureus.
By repeatedly sampling bacteria from the surviving colonies, and re-challenging each new culture with fresh surfaces,
the authors demonstrated that the bacteria did not develop resistance over the course of 11 exposures.
Figure 2 No resistance developed for quaternary
ammonium surfaces. A series of bactericidal activity assays
for amino-glass slides covalently coupled with Nhexyl,methyl-PEI against airborne S.aureus (A) and E. coli
(B). In each assay, the bacterial population originated from a
single colony of surviving bacteria from the preceding assay.
From Milovic et al. Biotechnology and Bioengineering.
2005;90(6):715-722.
Like many of the PQAs, polyethyleneimine is formed via relatively uncontrolled methods, that generate a wide range
of different molecular weights as well as branching topologies. A number of groups have used controlled radical
polymerization to produce PQAs that are monodisperse and with uniform molecular architectures. Some researchers
have used graft-to immobilization of these uniform and controlled block polymers created by ATRP[26]. The polymers
contained surface grafting regions as well as dimethyl amine regions. The surface density of the immobilized PQA was
controlled via the polymer solution concentration, immersion time and molecular architecture. They were able to
correlate the surface charge density with their observed efficacy. In the previous work, the initiator for ATRP was free
in solution when the polymer was synthesized; however, by immobilizing the initator to the substrate the same
controlled polymer morphology could be achieved, and with some added control over the polymer chain density. The
same group coated glass substrates with ATRP initiators for the controlled polymerization of
dimethylaminoethylmethacrylate[24]. Subsequent quaternization with alkylhalides formed the immobilized PQAs. By
controlling the surface density of initiators and the polymerization reaction time, they were able to independently
control the polymer density and polymer chainlength respectively. This enabled systematic evaluation of key
parameters without concern for elution of polymers. They concluded that the key operational parameter in efficacy was
the surface charge density, and not necessarily the polymer length. This has mechanistic implications, which they
explored and compared to other literature. According to this work, as long as the PQA surface charge density is greater
than 5x1015 charges/cm2 the film will be efficacious against E. coli, which coincidentally also has a surface charge
Of course both of these studies fully
density of 1014-1015 charges/cm2 depending on the growth stage of the cell.
characterized their substrates via surface analytical tools, and an interesting observation came when comparing the
efficacies of graft-to and graft-from surfaces with comparable densities of QA groups, the graft-to surfaces were more
efficacious. The authors hypothesize that this is due to the observed heterogeneity in the graft-to films, resulting in
local regions of higher relative QA densities. By using micropatterning they were able to generate areas of the substrate
where agent was immobilized directly next to areas free of the AMA[26]. This enabled their live-dead stain images to
spatially differentiate between kill over the immobilized agent from kill in neighboring unmodified areas (less than a
few microns away). This provides a highly credible method for stating that the agent does not kill by elution, and is
essentially a microscopic version of the ZOI test, but instead of being under growth conditions, it is under the more
relevant test conditions. This paper makes frequent connection between interesting mechanistically relevant molecular
properties of the film and the efficacy; for example, the number of QA groups needed to kill a bacterium, in this case
1010QA/bacterium.
©FORMATEX 2011
87
Science against microbial pathogens: communicating current research and technological advances
______________________________________________________________________________
A. Méndez-Vilas (Ed.)
Figure 3 Biocidal dependence on surface charge density.
The biocidal efficacy is related to the surface charge density
of QA groups, as probed by charge coupled fluorescence
assays. The grafting-to surfaces gave higher efficacy at
apparently lower densities, and this was hypothesized to result
from film heterogeneity, resulting in small areas with higher
charge densities.
From Huang et al. Langmuir.
2008;24(13):6785-6795.
Aromatic quaternary ammonium polymers
Of the aromatic PQAs, none is more studied than polyvinylpyridinium and the quaternized derivatives[32]. Given
nature of the pyridinium nitrogen, alkylation with a single group renders it quaternized. Researchers have focused on
the nature of the sidechain, it’s length and chemistry[21]. The authors found an interesting relationship between chain
length and efficacy, in that there was an optimal length of the N-alkylated group, with hexyl ammonium quats having
the greatest efficacy relative to the longer or shorter chainlengths. Other groups performed charge measurements on
quaternized PVP films and reported rapid kill in less than 10 minutes with live-dead staining[25]. They also performed
some interesting analysis of the bacterial state. They found that the different cellular states (low or high cell division
conditions) required a different surface charge density (1014 N+/cm2) for E. coli and S. epidermidis in the low cell
division state, but 1013 and 1012 in the high division conditions respectively. Given that they observed efficacy for films
with thicknesses of 2nm, they proposed that their data supports an ion exchange mechanism of efficacy[21,87,88]. PVP
AMAs have also been synthesized from a host of surfaces e.g., cellulose, polyethyleneterephthalate (PET) and
electrospun polyurethanes[33,34]. In each case, the polymerization was initiated from plasma activated surfaces. These
PVP modified surfaces were then quaternized with hexylbromide, the optimum length reported above. Yao et al.
challenged the electrospun membranes with S. aureus and E. coli by an immersion method. The modified membranes
showed a higher propensity for cell death for the S. aureus than the E. coli, with the former having a 5 log reduction in
viable cell count after 4 hours, and the latter having an LRV of only 3. SEM images of the membranes showed the
absence of intact cellular material on the treated samples, therefore the reduction was not simply due to selective
adhesion to the sample.
Figure 4 Optimum chainlength. A series of different nalkylhalides were used to quaternize surface bound
pyridinium polymers. The efficacy depended on the length of
the alkylhalide, with the optimum length corresponding to
hexyl bromide. From Tiller et al. Proc. Natl. Acad. Sci.
U.S.A. 2001;98(11):5981-5985.
The common form for these quaternary ammonium compounds include not only the quaternary ammonium group,
but also a hydrophobic side chain. The historical hypothesis is that the length and hydrophobicity of the side chain
functions to interact with the bacterial membrane, thereby assisting with the disruption of the membrane. As such most
of the sidechains have been aliphatic hydrocarbons; however, some work has explored alternative hydrophobic groups,
notably perfluorinated side groups[79]. This work compared the use of fluorinated (F(CF2)8(CH2)6Br) and
nonfluorinated (H(CH2)6Br) molecules for the quaternization of PVP coated styrene ethylene butylene styrene (SEBS).
Their primary observation was that fluorinated side chains improved efficacy, but they also observed high levels of
quaternization led to a reduced efficacy.
88
©FORMATEX 2011
Science against microbial pathogens: communicating current research and technological advances
_______________________________________________________________________________
A. Méndez-Vilas (Ed.)
Amine containing molecules and polymers
Based on modes of action that involve molecular charge, the less substituted amines should be efficacious as well[41].
Under normal pH conditions primary, secondary and tertiary amines will be protonated, and therefore positively
charged. In an interesting series of experiments, Lichter and Rubner studied the efficacy of LbL films composed of
alternating layers of the positively charged primary amine polyallylaminehydrochloride (PAH) and the negatively
charged polystyrenesulfonate (PSS)[39]. As with the alternate work described above[26], they observed that the
efficacy depended primarily upon generating a surface with sufficient positive charge density. Using plasma methods,
dimethylaminomethylstyrene was polymerized on textiles[38]. They found that a critical added mass was needed to
attain substantial antimicrobial efficacy, consistent with previous work that observed critical charge density. Although
based on the method of formation, these films are expected to be composed of many loosly bound polymers. Meaning
that thicker films might release more free molecules thereby leading to better efficacy via elution mechanisms. A 6-7
log reduction in microbe activity relative to unmodified controls was observed, and they used zone of inhibition tests to
confirm that their fabric swath was not eluting. Still other groups created surface initiated ATRP growth of
polymethacrylates with butyl and ethylamine groups[40]. Film thicknesses ranging from 3nm to 70nm all gave rapid
and complete kill; furthermore, dilution of the surface initiators from 100 to 1% had virtually no impact on the
performance of the film. Unfortunately the films lost antimicrobial activity with repeated exposure/rinsing cycles.
They observed massive kill within five minutes inoculation contact time. As with many researchers they used ZOI as a
method to prove that the agent was not leaching.
Figure 5 Primary amines can be efficacious. Consistent
with the observations above, primary amines can be
efficacious, if the surfaces are charged. In the case of primary
amines, this can be achieved via protonation. These layer-bylayer experiments showed that the efficacy could be switched
by simply changing the state of the film, via rinsing in pH
solutions above or below the pHa of the film. From Lichter &
Rubner Langmuir. 2009;25(13):7686-7694
Chlorhexidine (CHX), polyhexamethylenebiguanide (PHMB), polyhexamethyleneguanide (PHMG), various
oligoguanides and other biguanides have long been recognized for their antimicrobial activity and low human
toxicity[4],[75]. As membrane distruptive agents, they may retain some efficacy when immobilized. Researchers have
immobilized biguanides by crosslinking to polyacrylic acid[89], electrospinning[90] as a polymer composite and by
polymerization of synthetic biguanide-acrylates[91]. In each case, analytical methods associated the biguanides with
the substrate, but the arguments against elution based efficacy were supported by at best ZOI testing.
Chitosan is an inexpensive, naturally occuring polymer that has shown efficacy in solution as an AMA and on
surfaces as an antifouling agent. It has been immobilized to fibrous substrates such as wool, cotton, pulp, etc, and these
investigations have almost exclusively used shake-flask and ZOI testing to demonstrate efficacy and immobilization.
typically combined immersion inoculation methods with ZOI to demonstrate efficacy and support the lack of elution.
In one study, polypropylene films were modified with medium molecular weight chitosan via standard coupling
chemistries[45]. The authors observed a 3-5 log reduction in the viable bacteria depending on the bacterium (E.coli or
B. subtilus). Another group coated PMMA substrates with chitosan and observed that the surfaces were antimicrobial.
By exploiting live-dead staining and time-lapse confocal fluorescence microscopy, they were able to visualize the realtime permeabilization (death) of the microbes as they approached and interacted with the surface[49].
Peptides
All organisms produce antimicrobial peptides[92],[93],[94], and the online antimicrobial peptide database has an
outstanding coverage of the known peptides that have been studied in the literature[95]. The database is an excellent
©FORMATEX 2011
89
Science against microbial pathogens: communicating current research and technological advances
______________________________________________________________________________
A. Méndez-Vilas (Ed.)
resource for anyone interested in AMPs. While the cost of AMPs will be prohibitive for many applications, there may
be some device applications where the quantity needed may be appropriate. The fraction of peptides in the database
that are known or suspected to be membrane disruptive is relatively short, including molecules such as, magainin I,
polymyxin B, defensins, apoprotinin, nisin, etc. The mode of action for membrane disruptive peptides suggests some
key structural properties, and some authors have correlated structure (charge, hydrophobicity and spatial structure) with
the antimicrobial performance[60]. In this work, they identified target peptides and verified that they were efficacious.
The key structure of the efficacious peptides was that they possessed amphipathic and cationic structures. One well
known AMP was immobilized to gold alkylthiolate monolayers, and these surfaces exhibited contact kill[56]. Still
other groups tethered MAG to the end of surface bound antifouling PEG chains. Unlike much of the AMP-surface
crosslinking work which reacts with amine groups on the peptide, these authors exploited a thiol group on the MAG to
orient peptide. Interestingly, they observed that even low immobilization densities were efficacious. The samples were
inoculated via immersion in suspensions of two Gram-positive bacteria (Listeria ivanovii and Bacillus cereus), lightly
rinsed and stained with a live-dead stain. The efficacy was assessed by confocal laser scanning microscopy of the
stained cells. The images demonstrated that some of the filamentous B. cereus and all of the L. ivanovii cells which
remained following rinsing were dead. In addition to MAG, another popular AMP is Polymyxin B (PMB), which has
been immobilized for biowarfare sensors[57]. Like MAG, immobilized PMB has demonstrated some efficacy, though
by rather non-standard testing method. In these cases, the authors examined a shift in the growth curve for bacteria in
the presence of the modified surface[58]. Unlike most leaching tests, these authors also used a novel field effect
transistor method to confirm a lack of leached PMB. Elastomers such as PDMS have also been modified with AMPs,
in an effort to attain antibiofilm properties[59].
Figure 6 Immobilized peptides show kill. The antimicrobial
peptide magainin I was immobilized to gold thiolate
monolayers with standard coupling chemistry. By using livedead staining, the control thiol surface without peptide (a) was
not efficacious, while the magainin I modified surface (b) was
efficacious.
From Humblot et al. Biomaterials.
2009;30(21):3503-3512.
In addition to the naturally occuring and synthetic peptides, some groups have been working on synthetic peptoids
(molecules that will not be vulnerable to proteolytic degradation. Patch and Barron give an excellent review of nonnatural peptidomimetic oligomers[63], and Statz and coworkers[65] examined the impact that surface bound peptide
mimetics have on E. coli adhesion. In this study, the authors immobilized three different peptoid sequences to titania
substrates, an antimicrobial peptoid, an antihemolysis/antifouling peptoid and a filler peptoid. Immobilization was
confirmed with an assortment of surface analytical tools, and the antimicrobial efficacy of the surfaces were determined
with fluorescence microscopy. Their fluorescence data agreed with the solution phase minimum inhibitory
concentration data for the free peptoids.
Enzymes
As with peptides, most organizms generate enzymes as a part of their antimicrobial strategy. Several of these naturally
occuring enzymes have been used as bacteriocidal and antibiofilm agents. Also like the peptides, these will likely have
limited applicability, due both the cost and the sensitivity to thermal and proteolytic degradation. Chitinases have been
mobilized against fungi, and proteases have also been applied against prions. Autolysins, are a group of enzymes
generated by bacteria for regulation of their own cell wall, and these are usually highly specific to the originating
bacteria. Some common antimicrobial enzymes (AMEs) include proteinase K, trypsin, subtilisin, protease A, papain,
umamizyme, dispersin B, neutrophil elastase, phospholipase A2 and of course lysozyme. Lysozyme has been
immobilized to fabrics such as cotton[51] and wool[50], as well as polymer substrates such as polymethylmethacrylate,
polyethylene, polypropylene, polystyrene[52],[53]. The elution of the lysozyme has sometimes been tracked with
90
©FORMATEX 2011
Science against microbial pathogens: communicating current research and technological advances
_______________________________________________________________________________
A. Méndez-Vilas (Ed.)
HPLC, and following extensive rinsing was determined to be negligible. There are sensitive assays that can detect trace
quantities of free enzyme. The antimicrobial efficacy of the immobilized enzyme was monitored in the same way that
the activity of the enzyme would be determined, via a UV absorbance assay for the lysis of Micrococcus lysodeikticus.
The authors found that the efficacy increased with the quantity of lysozyme immobilized in their PVA matrix. Enzymes
have also been used to provide antifouling capabilities[96].
Efficacy testing
One of the most challenging components to the assessment of immobilized antimicroial agents is the efficacy test. The
methods are not inherently difficult to perform, in fact many of the test methods are relatively simple and readily
implemented. The difficulty centers on the interpretation of the results, which are directly related to the choice of
method. Method selection and implementation requires the synthesis of not only microbiology but also surface science,
fluid dynamics and assorted chemical engineering fields. The test methods that have been used to date can be grouped
into two different categories: (1) growth-based amplification/inhibition; and (2) live-dead staining. Given that these
studies are for immobilized agents, the methods are necessarily heterogeneous, with a solid phase on which the agent is
immobilized and a liquid phase. The immobilized substrate can be inoculated with the microbes in a range of different
manners, including immersion of the sample within a large volume of inoculum, direct contact of a small droplet onto
the surface of the sample, aerosolization of microbes onto the surface, as well as other interesting variants. When
choosing the method, and in anticipation of results and interpretation, it is useful to consider mass transport. Mass
transport of the microbe to and from the substrate as well as transport of potentially eluting agents from the substrate.
These processes will depend upon the method chosen, and open a range of questions that directly impact the
interpretation of the results. For example, does the eluted agent develop a concentration gradient at the surface, perhaps
within a stagnant layer? What is the dimension of that layer and how does the concentration of the agent in that layer
compare to the bulk concentration outside the stagnant layer? If cells enter this region, and perhaps adhere to the
surface, then they will likely experience concentrations much greater than the bulk, but how much more will depend
upon numerous variables.
Zone of inhibition
Zone of inhibition (ZOI) methods involve placing an AMA loaded substrate in contact with a growth media loaded with
bacteria. As the AMA elutes from the substrate into the media a zone may be observed where the concentration
exceeds the MIC or critical concentration for that AMA. The size of the zone is related to the diffusion constant for the
AMA in the media as well as the total amount of agent that is available to diffuse[98-100]. For truly immobilized
AMAs the quantity of material is likely to be severely limited, and can be estimated by knowledge of the approximate
surface area of the part. A brief calculation of the expected zone is recommended for anyone using this method to
conclude that the agent is immobilized. It may be that even if all of the AMA molecules eluted from the surface, the
volume corresponding to the critical concentration may correspond to an undetectably small zone. It is also important to
compare similar environments. The growth media used for ZOI may be inappropriate when the enumeration testing is
performed in a much cleaner saline suspension, which may contain fewer potential interferents. The interferences will
generate a higher MIC and will reduce the dimensions of the zone by an amount that depends upon the extent of
interference. This might be especially relevant for AMAs that exploit charge-based interactions, as the nutrient rich
media common to ZOI experiments contain proteins and polysaccharides that may contain ionized groups. Together
these factors can negate the value of ZOI testing for samples where the agent was intended to be immobilized.
Figure 7 Zone of inhibition. The zone of inhibition test is a
visual test where bacterial growth is inhibited from a region
around a sample. The sample is in or on growth media, and
the zone represents the diffusion volume wherein the agent
has eluted with concentrations adequate to inhibit growth
(MIC) in the growth medium. From Lee et al. Langmuir.
2005;21(21):9651-9659.
©FORMATEX 2011
91
Science against microbial pathogens: communicating current research and technological advances
______________________________________________________________________________
A. Méndez-Vilas (Ed.)
Immersive inoculation
The most common tests used for immobilized AMAs are the immersive inoculation methods, especially the shake flask
method ASTM E2149 which has been specifically called out for use with immobilized agents[101]. These methods
involve the complete immersion of a sample into an inoculum solution which is agitated for a specified time. After the
inoculation time, an aliquot of the microbial suspension is sampled and the viability is tested, usually via growth based
amplification. Factors such as cell adhesion and elution of the agent can dramatically impact the interpretation of the
results, as will be discussed below. Since the method actually measures the viability of the cells that remain suspended,
as viable cells are removed from the suspension by adhesion to the sample, they are counted as dead cells. A control
sample is used for comparison to the active sample, and while the control and active samples are often made of the
same substrate material (size, surface area, roughness, etc.), the nature of chemical modification, necessitates that the
sample and control will have very different chemistries. As shown above, most of the antimicrobial agents are positive,
and frequently there is a correlation between the efficacy and the positive charge density on the surface. This charge
difference could have a dramatic impact on the cell-surface adhesion, since many of the bacterial cell surfaces contain
negatively charged polymers.
In an effort to prove that the leaching of the AMA is irrelevant to the test method, some researchers have immersed
the sample in an inoculum-free solution, and then inoculated this solution in absence of the sample[23]. The lack of
efficacy of this post-sample-contact solution was used to rule out leaching as a factor for kill. Mass transport of the
agent from a surface is key to interpreting these results[97], and while the theory is conceptually rather simple and wellknown, a predictive understanding can be very complicated in a real-world system. Nonetheless, a thought experiment
that examines the various possible outcomes may be instructive. Firstly, and as expected, if the AMA is truly
immobilized, then the solution will be nonefficacious. Furthermore, if the AMA molecules rapidly elute from a part,
then the concentration in the bulk will rapidly approach a limiting value which may exceed the MBC for this solution.
If it does then it will be detected as efficacious, and the conclusion will be elution. However, if the bulk concentration
is inadequate to kill (perhaps due to dilution of a limited supply of AMA) then the conclusion will be that the agent does
not elute at a level needed to kill the microbes, when in fact it did elute, but it was diluted below the critical
concentration in that volume. These are all reasonable conclusions; however, an interesting outcome of this last
condition may occur when the elution rate is comparable to or less than the rate at which the cells sample the surface. In
this case, the planktonic cells approach a surface which is still eluting agent, and as the microbes do so, they will
experience concentrations in excess of the bulk concentration, and the microbes may die as a result, eventhough the
bulk solution remains far below the critical value (even at the end of the experiment). The conclusion of this would be
that the surface killed but not by elution, since the bulk solution concentration remains non-biocidal. This
oversimplified thought experiment can be used as a guide, but the reality is expected to be significantly more
complicated. This thought experiment does not contradict previous real experiments, but it does suggests that a
comprehensive understanding of the mechanism may benefit from a more critical tool capable of discriminating
between the immobilized and leachable kill. Perhaps some microscopic imaging methods near the surface under test
conditions comparable those of the shake flask method would provide insight.
Direct inoculation
Several groups have been using direct inoculation methods which place a small droplet of inoculum directly in contact
with the active surface. Two commonly cited methods have been developed by the Japanese Standards Association and
the International Standards Organization, JIS-Z-2801 and ISO 22196 respectively. Although these standard methods are
not explicitly designed for use with immobilized agents[102], it has nonetheless become common practice to apply
these direct methods to systems with purportedly immobilized agents. This method involves placing a small droplet (10100L) of inoculum directly on the surface of the sample, followed by placing a film on top of the droplet allowing
capillary forces to draw the surfaces together, thereby spreading the droplet across the surface. Following the requisite
inoculation time, the entire assembly (both surfaces and the captive liquid) is agitated and the released cells are typically
enumerated as CFUs. This coverslip, which is not usually antimicrobial, can adhere cells from the inoculum. It is not
uncommon to see direct inoculation results for ostensibly immobilized agents with log reduction values on the order of
3 to 8. While bacterial adhesion to the coverslip will depend upon the bacterial strain, some groups have observed 1025% of some strains of S. aureus and E. coli strongly adhere to the coverslip. If even 10% of the inoculum adhere to
the coverslip, and if 10% of these cells are recovered for enumeration, then the log reduction value (LRV) is expected to
be limited to less than 2. Therefore, high LRVs for direct inoculation methods are seemingly in contradiction to the
immobilized nature of the agent. It should be noted that the sample-coverslip separation is on the order of 5-25
microns, and that diffusion across this distance would be difficult to observe with ZOI, none the less ZOI is commonly
used in conjunction with direct methods to demonstrate that the agent is immobilized. The coverslip adhesion can be
avoided by excluding the coverslip, thereby providing the cells with only the sample surface. In summary, extra
attention should be paid to these kinds of enumeration based direct innoculation methods. This is especially true if the
test generates high LRVs, as this might be a good indication that there was elution.
92
©FORMATEX 2011
Science against microbial pathogens: communicating current research and technological advances
_______________________________________________________________________________
A. Méndez-Vilas (Ed.)
Surface growth methods
Whatever the means of inoculating the sample, the viability of microbes on the sample surface may be tested via taking
the surface and placing it in direct contact with a semi-solid nutrient rich media. Under appropriate conditions the
viable cells on the sample will grow into colonies that can then be enumerated. These methods amplify the viable cells
to visible colonies; however, they cannot be diluted, as they are attached to the surface, and so neighboring microbes
that are positioned within a set distance may lead to a single visible colony. Nonetheless, this semi-quantitative method
can be used to screen surfaces that are efficacious from those that are not. While any method of inoculation may be
used, it is most common to use this test method for aerosolization based inoculation experiments, wherein a thin film of
pathogens is applied across a surface. The bacteria are sometimes dried in place and sometimes kept humidified.
Following a specified inoculation time, the activated surfaces and controls are then used for growth based amplification.
These test methods are excellent methods to emulate ambient contamination of surfaces and the corresponding
antimicrobial efficacy of the surface; however, as it pertains to discrimination between immobilized and elutable agents,
there are a few points to consider. These methods place the bacteria in very close contact with the AMA coated
substrate. Even when the bacteria remain partially humidified, the volume of fluid in contact with the AMA coated
substrate is extremely small. Furthermore, if all of the fluid between the bacteria and the surface was removed, the
bacterial surface is still in direct contact with the surface, and diffusion of trace non-immobilized AMAs can still occur.
In this geometry, the impact of eluted agents is likely to be be greatly amplified, as compared even with the direct
inoculation methods above. By way of a very coarse example, suppose that a 1cm2 sample is inoculated, and suppose
that the same sample elutes enough free AMA into a 10mL solution to generate 1/1000 of the minimum bactericidal
concentration (MBC). Limited by the concentration at the source and allowing sufficient time for equilibration, the
concentration produced by the same amount of material released into the constrained volume of a thin film would be
higher. In the case of the direct inoculation methods, a 10m thick film would result in a concentration of 10 MBC.
For aerosol based methods, the fluid layer between the cells and the substrate is much thinner, (say 100nm) the relative
concentration may approach 1000MBC. Of course, 100nm is probably thicker than expected if the cell is in direct
contact with the surface, and so higher concentrations are plausible, with a real limit being the amount of free agent and
the solubility of the agent in that fluid/extracellular layer. This simplistic back-of-the-envelope calculation simply
highlights the potential amplification of trace elutables on the activity of the AMA modified surfaces. To the extent that
the mobility of the AMA is of interest, a more detailed calculation or test would be needed to assess the impact that
these potential elutables would have on a real system.
Fluorescent and luminescent methods
There are a number of semiquantitative methods that exploit luminsecence to detect the viability of microbes. The most
common of these methods use commercially available live-dead stain kits, where the stains probe various properties of
the microbe such as membrane permeablity, metabolic activity, etc.[106,107] These kinds of fluorescent stains can be
used with fluorescent microscopy, as well as flow cytometry.[108,109] One conceptually simple live-dead staining
technique has demonstrated the ability to determine whether the antimicrobial agent kills cells at the surface or at a
distance.[110] The method uses a direct inoculation method with spacers to separate an iAMA surface from a control
coverslip surface. The method generates three populations of bacteria that can be compared: (1) those at the control
surface, (2) those at the test iAMA surface and (3) those freely floating in the solution. Comparison of the bacterial
fluorescence in the three populations can provide insight as to whether the agent acts only at the substrate or is able to
affect the control bacteria. The process could be extended to include alternate fluorophores, thereby probing metabolic
activity or other properties of the microbes. As with the other test methods, live-dead staining has many limitations.
Firstly, it has a limited dynamic range (usually from 5-95 percent compared to enumeration methods that can vary over
several orders of magnitude). The live-dead staining can depend upon the bacterial species, the strain, or the medium.
In the case of immobilized surfaces, the stains can sometimes interact with the substrate producing high fluorescent
backgrounds, thereby swamping the bacterial signal. Bacterial adhesion to the surfaces is discussed in detail in the
following chapter.
Bioluminescence has also been exploited for testing antimicrobial properties of devices, though usually it is used for
biofouling and biofilm experiments[60,111]. Typically, these experiments genetically engineered lux-reporter strains,
and the bioluminescence is a measure of the cellular respiration. Following the initial transfection of the bacteria with a
gene for bioluminescent process, these bacteria are handeled in a similar manner to other bacterial strains. In principle,
this method could be applied to any bacterial strain to create a lux-version of that strain. However, once created this is a
new strain, and some of it’s energetics and architecture will be devoted to maintenance of the bioluminescence,
potentially impacting the ability to compete and defend against antimicrobial therapies.
©FORMATEX 2011
93
Science against microbial pathogens: communicating current research and technological advances
______________________________________________________________________________
A. Méndez-Vilas (Ed.)
Critical Discussion and Conclusions
Without a doubt, one of the most detrimental activities to the proper interpretation of the immobilized AMA efficacy
has been the use of ZOI testing as a rubber stamp verification that the AMA is not eluting. While these methods are
good for identifying massive elution from loaded pads, the numbers do not support ZOI application to monolayer or
even multilayer modified samples. The next most hazardous activity has been the use of JIS style direct inoculation
methods to obtain quantitative efficacy for agents that are presumed immobilized as a result of ZOI data. These direct
measurements sometimes provide extremely high log reduction values, and it has been suggested in the literature, that
these high values correspond to an immobilized agent that was exceptionally efficacious eventhough bound to the
substrate. Depending upon cell adhesion and the experimental details these results may be accurate, but there may also
be a serious flaw, which was outlined above, and the data may in fact support eluting efficacy. These two factors may
have misdirected researchers away from truly immobilized efficacious agents, because of apparently low efficacies and
toward agents whose efficacy is bolstered by the freedom of diffusion from elution.
As with any test method, the perceived efficacy will be convoluted with the capabilities of the test method, and since
many of the above methods exploit the growth of viable microbes in a growth medium, these growth based methods
will be limited when the microbe is viable but non-culturable (VBNC)[103-105]. If the microbe has been put into a
VBNC state, and resists growth in the medium, then it will appear to have been killed by the agent, while it actually
remains viable, awaiting on an appropriate trigger or medium to reactivate. The assessment of an immobilized AMCs
ability to kill VBNCs will be strengthened by the development of methods capable of monitoring the activity of
VBNC’s. This field is relatively new, and new VBNC appropriate methods will likely require detailed understanding of
particular species, strain or even phenotypic properties.
One of the key potential drawbacks for immobilized AMAs comes from the fact that the surface may become fouled
by the very bacteria that the surface kills. This is expected to be especially true for the surfaces designed to operate by
the positive charge density threshold mechanism of action. These are expected to adhere the negatively charged
bacteria and upon inactivation, the negatively charged cellular material will likely remain to obscure the surface,
thereby inactivating the efficacy in that region. Furthermore, low charge density regions that might allow live cells to
adhere can act as islands which may propagate, and grow colonies on top of a monolayer of dead cells. This is not a
fatal flaw for iAMAs, but is a key limitation, that should be considered when evaluating the immobilized agent for a
realistic application.
One other interesting limit to immobilized AMA is the expected upper limit for efficacy. Unlike eluting agents,
immobilized agents will require that the microbe comes into close contact with the surface, and while surfaces may be
regenerated (via a cleaning protocol), it seems reasonable that the efficacy may be limited by the total surface area of
the agent normalized by the size of the microbe. In one example researchers challenged their samples with increasing
inoculum, and observed an interesting limit to the efficacy of their surfaces (~1x108 E. coli/cm2). This value is
approximately equal the closest packed surface coverage of these bacteria on a surface. Testing to failure like this can
be very informative, and where appropriate, it is recommended to provide further mechanistic insight into the test
method and mode of action.
As with many technologies, the performance of materials are sometimes oversold, with best-case instead of typical
conditions being presented. From discussions with numerous other researchers in the field, it is clear that a fair fraction
of published work is very difficult to reproduce. This does not implicate the published work, so much as perhaps
highlights the number of variables that may change from sample to sample, lab to lab, strain to strain, slant to slant, etc.
It should; however, caution the reader and researcher to plan for additional resources and testing when embarking on an
iAMA project.
A multitude of strategies and materials have been used to create surfaces with immobilized antimicrobial agents.
Emerging directions for the agents and the agent properties include a focus on combining antifouling with antimicrobial
properties. Alternate promising directions include responsive or smart materials capable of switching from antifouling
to antimicrobial when stimulated by the presence of microbes. In a closely related direction, some researchers have
focused on microstructured surfaces to minimize biofilm formation. Future developments of this textured aspect might
generate added functionality. As new materials are added to surfaces for biomedical devices, the cytotoxicity will also
be of interest; however, given that the agents are immobilized, the toxicity is expected to be less important than for the
corresponding biomedical devices with leaching antimicrobial agents.
Depending upon the stringency of the immobilized criteria, there may be many examples of immobilized agents or
very few. This pursuit is complicated by testing method appropriateness, bacterial species/strains, resistance, and
simple microbe surface interactions. This review has highlighted the efforts to date with regards to immobilization of
antimicrobial agents, and was intended to cast some critical light on the appropriateness of the efficacy testing as it
pertains to truly immobilized agents.
94
©FORMATEX 2011
Science against microbial pathogens: communicating current research and technological advances
_______________________________________________________________________________
A. Méndez-Vilas (Ed.)
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
Klevens R, Edwards J, Richards C, et al. Estimating health care-associated infections and deaths in US hospitals, 2002. Public
Health Reports. 2007;122(2):160-166.
Gabriel G, Som A, Madkour A, Eren T, Tew G. Infectious disease: Connecting innate immunity to biocidal polymers.
Materials Science & Engineering R-reports. 2007;57(1-6):28-64.
McDonnell GE, Ph.D. Antisepsis, Disinfection, and Sterilization: Types, Action, and Resistance. 1st ed. ASM Press; 2007.
Milstone A, Passaretti C, Perl T. Chlorhexidine: Expanding the armamentarium for infection control and prevention. Clinical
Infectious Diseases. 2008;46(2):274-281.
Rai M, Yadav A, Gade A. Silver nanoparticles as a new generation of antimicrobials. Biotechnology Advances.
January;27(1):76-83.
Monteiro DR, Gorup LF, Takamiya AS, et al. The growing importance of materials that prevent microbial adhesion:
antimicrobial effect of medical devices containing silver. International Journal of Antimicrobial Agents. 2009;34(2):103-110.
Zilberman M, Elsner J. Antibiotic-eluting medical devices for various applications. Journal of Controlled Release.
2008;130(3):202-215.
Vasilev K, Cook J, Griesser H. Antibacterial surfaces for biomedical devices. Expert Review of Medical Devices.
2009;6(5):553-567.
Charnley M, Textor M, Acikgoz C. Designed polymer structures with antifouling-antimicrobial properties. Reactive &
Functional Polymers. 2011;71(3):329-334.
Gettings RL, White WC. Formation of polymeric antimicrobial surfaces from organofunctional silanes. Polym. Mater. Sci. Eng.
1987;57:181-5.
Isquith AJ, Abbott EA, Walters PA. Surface-Bonded Antimicrobial Activity of an Organosilicon Quaternary Ammonium
Chloride. Appl Microbiol. 1972;24(6):859-863.
Osinska-Jaroszuk M, Ginalska G, Belcarz A, Uryniak A. Vascular Prostheses with Covalently Bound Gentamicin and
Amikacin Reveal Superior Antibacterial Properties than Silver-impregnated Ones - An In Vitro Study. European Journal of
Vascular and Endovascular Surgery. 2009;38(6):697-706.
Namba N, Yoshida Y, Nagaoka N, et al. Antibacterial effect of bactericide immobilized in resin matrix. Dental Materials.
2009;25(4):424-430.
Andresen M, Stenstad P, Moretro T, et al. Nonleaching Antimicrobial Films Prepared from Surface-Modified Microfibrillated
Cellulose. Biomacromolecules. 2007;8(7):2149-2155.
Nikawa H, Ishida K, Hamada T, et al. Immobilization of octadecyl ammonium chloride on the surface of titanium and its effect
on microbial colonization in vitro. Dent. Mater. J. 2005;24(4):570-582.
Gottenbos B, van der Mei HC, Klatter F, Nieuwenhuis P, Busscher HJ. In vitro and in vivo antimicrobial activity of covalently
coupled quaternary ammonium silane coatings on silicone rubber. Biomaterials. 2002;23(6):1417-1423.
Li Z, Lee D, Sheng X, Cohen RE, Rubner MF. Two-Level Antibacterial Coating with Both Release-Killing and Contact-Killing
Capabilities. Langmuir. 2006;22(24):9820-9823.
Harney M, Pant R, Fulmer P, Wynne J. Surface Self-Concentrating Amphiphilic Quaternary Ammonium Biocides as Coating
Additives. Acs Applied Materials & Interfaces. 2009;1(1):39-41.
Hu FX, Neoh KG, Cen L, Kang ET. Antibacterial and antifungal efficacy of surface functionalized polymeric beads in repeated
applications. Biotechnol. Bioeng. 2005;89(4):474-484.
Kurt P, Wood L, Ohman D, Wynne K. Highly effective contact antimicrobial surfaces via polymer surface modifiers.
Langmuir. 2007;23(9):4719-4723.
Tiller JC, Liao CJ, Lewis K, Klibanov AM. Designing surfaces that kill bacteria on contact. Proc. Natl. Acad. Sci. U.S.A.
2001;98(11):5981-5985.
Lewis K, Klibanov A. Surpassing nature: rational design of sterile-surface materials. Trends in Biotechnology. 2005;23(7):343348.
Haldar J, An D, de Cienfuegos L, Chen J, Klibanov A. Polymeric coatings that inactivate both influenza virus and pathogenic
bacteria. Proceedings of the National Academy of Sciences of the United States Of. 2006;103(47):17667-17671.
Murata H, Koepsel R, Matyjaszewski K, Russell A. Permanent, non-leaching antibacterial surfaces - 2: How high density
cationic surfaces kill bacterial cells. Biomaterials. 2007;28(32):4870-4879.
Kügler R, Bouloussa O, Rondelez F. Evidence of a charge-density threshold for optimum efficiency of biocidal cationic
surfaces. Microbiology (Reading, Engl.). 2005;151(Pt 5):1341-1348.
Huang J, Koepsel RR, Murata H, et al. Nonleaching antibacterial glass surfaces via “Grafting Onto”: the effect of the number of
quaternary ammonium groups on biocidal activity. Langmuir. 2008;24(13):6785-6795.
Huang J, Murata H, Koepsel R, Russell A, Matyjaszewski K. Antibacterial polypropylene via surface-initiated atom transfer
radical polymerization. Biomacromolecules. 2007;8(5):1396-1399.
Lee S, Koepsel R, Morley S, et al. Permanent, nonleaching antibacterial surfaces. 1. Synthesis by atom transfer radical
polymerization. Biomacromolecules. 2004;5(3):877-882.
Ravikumar T, Murata H, Koepsel R, Russell A. Surface-active antifungal polyquaternary amine. Biomacromolecules.
2006;7(10):2762-2769.
Milovic N, Wang J, Lewis K, Klibanov A. Immobilized N-alkylated polyethylenimine avidly kills bacteria by rupturing cell
membranes with no resistance developed. Biotechnology and Bioengineering. 2005;90(6):715-722.
Wong S, Li Q, Veselinovic J, et al. Bactericidal and virucidal ultrathin films assembled layer by layer from polycationic Nalkylated polyethylenimines and polyanions. Biomaterials. 2010;31(14):4079-4087.
Sharma S, Chauhan G, Gupta R, Ahn J. Tuning anti-microbial activity of poly(4-vinyl 2-hydroxyethyl pyridinium) chloride by
anion exchange reactions. Journal of Materials Science-materials in Medicine. 2010;21(2):717-724.
©FORMATEX 2011
95
Science against microbial pathogens: communicating current research and technological advances
______________________________________________________________________________
A. Méndez-Vilas (Ed.)
[33] Cen L, Neoh KG, Kang ET. Surface Functionalization Technique for Conferring Antibacterial Properties to Polymeric and
Cellulosic Surfaces. Langmuir. 2003;19(24):10295-10303.
[34] Yao C, Li X, Neoh K, Shi Z, Kang E. Surface modification and antibacterial activity of electrospun polyurethane fibrous
membranes with quaternary ammonium moieties. Journal of Membrane Science. 2008;320(1-2):259-267.
[35] Lin J, Qiu S, Lewis K, Klibanov A. Bactericidal properties of flat surfaces and nanoparticles derivatized with alkylated
polyethylenimines. Biotechnology Progress. 2002;18(5):1082-1086.
[36] Martin T, Chan K, Gleason K. Combinatorial initiated chemical vapor deposition (iCVD) for polymer thin film discovery. Thin
Solid Films. 2008;516(5):681-683.
[37] Martin T, Kooi S, Chang S, Sedransk K, Gleason K. Initiated chemical vapor deposition of antimicrobial polymer coatings.
Biomaterials. 2007;28(6):909-915.
[38] Martin T, Lau K, Chan K, et al. Initiated chemical vapor deposition (iCVD) of polymeric nanocoatings. Surface & Coatings
Technology. 2007;201(22-23):9400-9405.
[39] Lichter JA, Rubner MF. Polyelectrolyte Multilayers with Intrinsic Antimicrobial Functionality: The Importance of Mobile
Polycations. Langmuir. 2009;25(13):7686-7694.
[40] Madkour A, Dabkowski J, Nusslein K, Tew G. Fast Disinfecting Antimicrobial Surfaces. Langmuir. 2009;25(2):1060-1067.
[41] Kuroda K, Caputo G, DeGrado W. The Role of Hydrophobicity in the Antimicrobial and Hemolytic Activities of
Polymethacrylate Derivatives. Chemistry-a European Journal. 2009;15(5):1123-1133.
[42] Park D, Wang J, Klibanov AM. One-step, painting-like coating procedures to make surfaces highly and permanently
bactericidal. Biotechnol. Prog. 2006;22(2):584-589.
[43] Alonso D, Gimeno M, Olayo R, et al. Cross-linking chitosan into UV-irradiated cellulose fibers for the preparation of
antimicrobial-finished textiles. Carbohydrate Polymers. 2009;77(3):536-543.
[44] Rabea E, Badawy M, Stevens C, Smagghe G, Steurbaut W. Chitosan as antimicrobial agent: Applications and mode of action.
Biomacromolecules. 2003;4(6):1457-1465.
[45] Vartiainen J, Ratto M, Tapper U, Paulussen S, Hurme E. Surface modification of atmospheric plasma activated BOPP by
immobilizing chitosan. Polymer Bulletin. 2005;54(4-5):343-352.
[46] Asadinezhad A, Novak I, Lehocky M, et al. Polysaccharides Coatings on Medical-Grade PVC: A Probe into Surface
Characteristics and the Extent of Bacterial Adhesion. Molecules. 2010;15(2):1007-1027.
[47] Busscher H, Engels E, Dijkstra R, van der Mei H. Influence of a chitosan on oral bacterial adhesion and growth in vitro.
European Journal of Oral Sciences. 2008;116(5):493-495.
[48] Lim S-H, Hudson SM. Application of a fiber-reactive chitosan derivative to cotton fabric as an antimicrobial textile finish.
Carbohydrate Polymers. 2004;56(2):227-234.
[49] Carlson RP, Taffs R, Davison WM, Stewart PS. Anti-biofilm properties of chitosan-coated surfaces. J Biomater Sci Polym Ed.
2008;19(8):1035-1046.
[50] Wang Q, Fan X, Hu Y, et al. Antibacterial functionalization of wool fabric via immobilizing lysozymes. Bioprocess and
Biosystems Engineering. 2009;32(5):633-639.
[51] Ibrahim N, Gouda M, El-Shafei A, Abdel-Fatah O. Antimicrobial activity of cotton fabrics containing immobilized enzymes.
Journal of Applied Polymer Science. 2007;104(3):1754-1761.
[52] Conte A, Buonocore G, Bevilacqua A, Sinigaglia M, Del Nobile M. Immobilization of lysozyme on polyvinylalcohol films for
active packaging applications. Journal of Food Protection. 2006;69(4):866-870.
[53] Conte A, Buonocore G, Sinigaglia M, Del Nobile M. Development of immobilized lysozyme based active film. Journal of
Food Engineering. 2007;78(3):741-745.
[54] Zhou Y, Lim L. Activation of Lactoperoxidase System in Milk by Glucose Oxidase Immobilized in Electrospun Polylactide
Microfibers. Journal of Food Science. 2009;74(2):C170-C176.
[55] Vartiainen J, Ratto M, Paulussen S. Antimicrobial activity of glucose oxidase-immobilized plasma-activated polypropylene
films. Packaging Technology and Science. 2005;18(5):243-251.
[56] Humblot V, Yala J, Thebault P, et al. The antibacterial activity of Magainin I immobilized onto mixed thiols Self-Assembled
Monolayers. Biomaterials. 2009;30(21):3503-3512.
[57] Kulagina N, Shaffer K, Anderson G, Ligler F, Taitt C. Antimicrobial peptide-based array for Escherichia coli and Salmonella
screening. Analytica Chimica Acta. 2006;575(1):9-15.
[58] Tzoris A, Hall EAH, Besselink G, Bergveld P. Testing the Durability of Polymyxin B Immobilization on a Polymer Showing
Antimicrobial Activity: A Novel Approach with the Ion-Step Method. Analytical Letters. 2003;36(9):1781-1803.
[59] De Prijck K, De Smet N, Rymarczyk-Machal M, et al. Candida albicans biofilm formation on peptide functionalized
polydimethylsiloxane. Biofouling. 2010;26(3):269-275.
[60] Hilpert K, Elliott M, Jenssen H, et al. Screening and Characterization of Surface-Tethered Cationic Peptides for Antimicrobial
Activity. Chemistry & Biology. 2009;16(1):58-69.
[61] Hilpert K, Volkmer-Engert R, Henklein P, Farmer S, Hancock R. A novel method for screening and characterization of small
surface-tethered cationic peptides for antimicrobial activity. Biopolymers. 2007;88(4):574-574.
[62] Onaizi S, Leong S. Tethering antimicrobial peptides: Current status and potential challenges. Biotechnology Advances.
2011;29(1):67-74.
[63] Patch J, Barron A. Mimicry of bioactive peptides via non-natural, sequence-specific peptidomimetic oligomers. Current
Opinion in Chemical Biology. 2002;6(6):872-877.
[64] Statz A, Kuang J, Ren C, et al. Experimental and theoretical investigation of chain length and surface coverage on fouling of
surface grafted polypeptoids. Biointerphases. 2009;4(2):FA22-FA32.
[65] Statz A, Park J, Chongsiriwatana N, Barron A, Messersmith P. Surface-immobilised antimicrobial peptoids. Biofouling.
2008;24(6):439-448.
[66] White WC, Lefler M. Medical textiles: versatile in use but vulnerable to microbial problems. Nonwoven Tech. Text.
2009;2(2):9-18.
96
©FORMATEX 2011
Science against microbial pathogens: communicating current research and technological advances
_______________________________________________________________________________
A. Méndez-Vilas (Ed.)
[67] Codling C, Maillard J, Russell A. Aspects of the antimicrobial mechanisms of action of a polyquaternium and an amidoamine.
Journal of Antimicrobial Chemotherapy. 2003;51(5):1153-1158.
[68] Bagheri M, Beyermann M, Dathe M. Immobilization Reduces the Activity of Surface-Bound Cationic Antimicrobial Peptides
with No Influence upon the Activity Spectrum. Antimicrobial Agents and Chemotherapy. 2009;53(3):1132-1141.
[69] Chawner JA, Gilbert P. Interaction of the bisbiguanides chlorhexidine and alexidine with phospholipid vesicles: evidence for
separate modes of action. J Appl Bacteriol. 1989;66(3):253-8.
[70] Chen H, Leung K, Thakur N, Tan A, Jack R. Distinguishing between different pathways of bilayer disruption by the related
antimicrobial peptides cecropin B, B1 and B3. European Journal of Biochemistry. 2003;270(5):911-920.
[71] Dietrich D, Xiao X, Dawson D, et al. Human alpha- and beta-Defensins Bind to Immobilized Adhesins from Porphyromonas
gingivalis. Infection and Immunity. 2008;76(12):5714-5720.
[72] Ding L, Chi E, Chemburu S, et al. Insight into the Mechanism of Antimicrobial Poly(phenylene ethynylene) Polyelectrolytes:
Interactions with Phosphatidylglycerol Lipid Membranes. Langmuir. 2009;25(24):13742-13751.
[73] Ding L, Chi E, Schanze K, Lopez G, Whitten D. Insight into the Mechanism of Antimicrobial Conjugated Polyelectrolytes:
Lipid Headgroup Charge and Membrane Fluidity Effects. Langmuir. 2010;26(8):5544-5550.
[74] Huang J, Koepsel R, Murata H, et al. Nonleaching antibacterial glass surfaces via “Grafting Onto”: The effect of the number of
quaternary ammonium groups on biocidal activity. Langmuir. 2008;24(13):6785-6795.
[75] Lim K, Kam P. Chlorhexidine - pharmacology and clinical applications. Anaesthesia and Intensive Care. 2008;36(4):502-512.
[76] Chopra I, Roberts M. Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of
Bacterial Resistance. Microbiol Mol Biol Rev. 2001;65(2):232-260.
[77] Hermanson GT. Bioconjugate Techniques, Second Edition. 2nd ed. Academic Press; 2008.
[78] Hein C, Liu X, Wang D. Click chemistry, a powerful tool for pharmaceutical sciences. Pharmaceutical Research.
2008;25(10):2216-2230.
[79] Krishnan S, Ward RJ, Hexemer A, et al. Surfaces of Fluorinated Pyridinium Block Copolymers with Enhanced Antibacterial
Activity. Langmuir. 2006;22(26):11255-11266.
[80] Lichter J, Van Vliet K, Rubner M. Design of Antibacterial Surfaces and Interfaces: Polyelectrolyte Multilayers as a
Multifunctional Platform. Macromolecules. 2009;42(22):8573-8586.
[81] Nagel J, Brauer M, Hupfer B, et al. Investigations on the reactive surface modification of polycarbonate by surface-reactive
injection molding. Journal of Applied Polymer Science. 2004;93(3):1186-1191.
[82] Barbey R, Lavanant L, Paripovic D, et al. Polymer Brushes via Surface-Initiated Controlled Radical Polymerization: Synthesis,
Characterization, Properties, and Applications. Chemical Reviews. 2009;109(11):5437-5527.
[83] Venkataraman S, Zhang Y, Liu L, Yang Y. Design, syntheses and evaluation of hemocompatible pegylated-antimicrobial
polymers with well-controlled molecular structures. Biomaterials. 2010;31(7):1751-1756.
[84] Baum M, Brittain W. Synthesis of polymer brushes on silicate substrates via reversible addition fragmentation chain transfer
technique. Macromolecules. 2002;35(3):610-615.
[85] Brinks M, Studer A. Polymer Brushes by Nitroxide-Mediated Polymerization. Macromolecular Rapid Communications.
2009;30(13):1043-1057.
[86] Fristrup C, Jankova K, Hvilsted S. Surface-initiated atom transfer radical polymerization-a technique to develop biofunctional
coatings. Soft Matter. 2009;5(23):4623-4634.
[87] Record MT, Anderson CF, Lohman TM. Thermodynamic Analysis of Ion Effects on the Binding and Conformational
Equilibria of Proteins and Nucleic Acids: The Roles of Ion Association or Release, Screening, and Ion Effects on Water
Activity. Quarterly Reviews of Biophysics. 1978;11(02):103-178.
[88] Park SY, Bruinsma RF, Gelbart WM. Spontaneous overcharging of macro-ion complexes. Europhys. Lett. 1999;46(4):454-460.
[89] Asadinezhad A, Novak I, Lehocky M, et al. An in vitro bacterial adhesion assessment of surface-modified medical-grade PVC.
Colloids and Surfaces B-biointerfaces. 2010;77(2):246-256.
[90] Chen L, Bromberg L, Hatton TA, Rutledge GC. Electrospun cellulose acetate fibers containing chlorhexidine as a bactericide.
Polymer. 2008;49(5):1266-1275.
[91] Guan Y, Xiao H, Sullivan H, Zheng A. Antimicrobial-modified sulfite pulps prepared by in situ copolymerization.
Carbohydrate Polymers. 2007;69(4):688-696.
[92] Martin E, Ganz T, Lehrer RI. Defensins and other endogenous peptide antibiotics of vertebrates. Journal of Leukocyte Biology.
1995;58:128-136.
[93] Yeaman M, Yount N. Mechanisms of Antimicrobial Action and Release. Pharmacology Reviews. 2003;55(1):27-55.
[94] Jenssen H, hamill P, Hancock R. Peptide antimicrobial agents. Clinical Microbiology Reviews. 2006;19(3):491-511.
[95] Wang Z, Wang G. APD: the Antimicrobial Peptide Database. Nucleic Acids Research. 32:D590-D592.
[96] Asuri P, Karajanagi SS, Kane RS, Dordick JS. Polymer–Nanotube–Enzyme Composites as Active Antifouling Films. Small.
2007;3(1):50-53.
[97] Crank J. The Mathematics of Diffusion. 2nd ed. Oxford University Press, USA; 1980.
[98] Drugeon HB, Juvin ME, Caillon J, Courtieu AL. Assessment of formulas for calculating critical concentration by the agar
diffusion method. Antimicrob. Agents Chemother. 1987;31(6):870-875.
[99] Lee D, Cohen RE, Rubner MF. Antibacterial properties of Ag nanoparticle loaded multilayers and formation of magnetically
directed antibacterial microparticles. Langmuir. 2005;21(21):9651-9659.
[100]Kavanagh F [Ed]. Analytical Microbiology. Academic Press; 1963.
[101]American Society for Testing & Materials. ASTM E 2149-01 Standard Test Method for Determining the Antimicrobial Activity
of Immobilized Antimicrobial Agents Under Dynamic Contact Conditions. West Conshohocken, PA 19428: American Society
for Testing & Materials; 2001.
[102]Japanese Standards Association. JIS Z 2801:2000 Antimicrobial products – Test for antimicrobial activity and efficacy. Tokyo,
Japan: Japanese Standards Association; 2000.
©FORMATEX 2011
97
Science against microbial pathogens: communicating current research and technological advances
______________________________________________________________________________
A. Méndez-Vilas (Ed.)
[103]Oliver J. Recent findings on the viable but nonculturable state in pathogenic bacteria. Fems Microbiology Reviews.
2010;34(4):415-425.
[104]Giao M, Wilks S, Azevedo N, Vieira M, Keevil C. Validation of SYTO 9/Propidium Iodide Uptake for Rapid Detection of
Viable but Noncultivable Legionella pneumophila. Microbial Ecology. 2009;58(1):56-62.
[105]Sachidanandham R, Gin K, Poh C. Monitoring of active but non-culturable bacterial cells by flow cytometry. Biotechnology
and Bioengineering. 2005;89(1):24-31.
[106]Stocks S. Mechanism and use of the commercially available viability stain, BacLight. Cytometry Part A. 2004;61A(2):189-195.
[107]Joux F, Lebaron P. Use of fluorescent probes to assess physiological functions of bacteria at single-cell level. Microbes Infect.
2000;2(12):1523-1535.
[108]Chau F, Lefort A, Fantin B. Interest and applications of flow cytometry in medical bacteriology. Antibiotiques. 2008;10(4):226231.
[109]Hoefel D, Grooby W, Monis P, Andrews S, Saint C. Enumeration of water-borne bacteria using viability assays and flow
cytometry: a comparison to culture-based techniques. Journal of Microbiological Methods. 2003;55(3):585-597.
[110]Green J, Bickner S, Carter P, et al. Antimicrobial Testing for Surface-Immobilized Agents With a Surface-Separated Live-Dead
Staining Method. Biotechnology and Bioengineering. 2011;108(1):231-236.
[111]Galluzzi L, Karp M. Whole cell strategies based on lux genes for high throughput applications toward new antimicrobials.
Combinatorial Chemistry & High Throughput Screening. 2006;9(7):501-514.
98
©FORMATEX 2011