MSC-03176; No of Pages 8
Materials Science and Engineering C xxx (2011) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C
journal homepage: www.elsevier.com/locate/msec
Antimicrobial activity of transition metal acid MoO3 prevents microbial growth on
material surfaces ☆,☆☆
Cordt Zollfrank a,⁎, Kai Gutbrod a, Peter Wechsler b, Josef Peter Guggenbichler c
a
b
c
University of Erlangen-Nuremberg, Department of Materials Science and Engineering 3-Glass and Ceramics, Martensstr. 5, D-91058 Erlangen, Germany
LEONI Kabel GmbH, Stieberstraße 5, D-91154 Roth, Germany
Laboratory for the Development of Healthcare Products, Leitweg 23, A-6345 Kössen, Austria
a r t i c l e
i n f o
Article history:
Received 10 May 2011
Received in revised form 22 August 2011
Accepted 22 September 2011
Available online xxxx
Keywords:
Antimicrobial materials
Transition metal acid
Molybdenum oxide
Antibacterial coating
Sol–gel technique
a b s t r a c t
Serious infectious complications of patients in healthcare settings are often transmitted by materials and
devices colonised by microorganisms (nosocomial infections). Current strategies to generate material surfaces with an antimicrobial activity suffer from the consumption of the antimicrobial agent and emerging
multidrug-resistant pathogens amongst others. Consequently, materials surfaces exhibiting a permanent antimicrobial activity without the risk of generating resistant microorganisms are desirable. This publication reports on
the extraordinary efficient antimicrobial properties of transition metal acids such as molybdic acid (H2MoO4),
which is based on molybdenum trioxide (MoO3). The modification of various materials (e.g. polymers, metals)
with MoO3 particles or sol–gel derived coatings showed that the modified materials surfaces were practically
free of microorganisms six hours after contamination with infectious agents. The antimicrobial activity is based
on the formation of an acidic surface deteriorating cell growth and proliferation. The application of transition
metal acids as antimicrobial surface agents is an innovative approach to prevent the dissemination of microorganisms in healthcare units and public environments.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Health care associated infections (nosocomial infections: NI) are the
fourth leading cause of disease and the most common complication
affecting hospitalised patients in addition to a minimum of 175,000
deaths every year in industrialised countries [1–2]. Reports from the US
indicate that NI account for 2 million infections and 90,000 preventable
deaths per year [3]. Transmission of pathogens and resulting biofilm
growth has also gained increasing importance in industrial applications
(e.g. cooling towers of power plants), water treatment and sanitation,
food packaging and public environments (e.g. public transportation).
An increasing number of highly vulnerable patients together with
emerging antibiotic-resistant microbes, especially methicillin-resistant
Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus
species (VRE) and Gram-negative microorganisms producing an extended spectrum of beta-lactamases (ESBL) are encountered [4–7]. The situation is aggravated by the fact that in the future there will be few new
antibiotics under development to compensate for the increasing resistance, because of the high costs of research and clinical testing as well
as regulatory problems [8–11]. In view of increasing antibiotic resistance,
☆ The authors declare no competing financial interests.
☆☆ The authors are grateful to Dr. Carolin Körner (WTM, University of ErlangenNuremberg) for the kind gift of the titanium rods.
⁎ Corresponding author. Tel.: + 49 9131 85 27560; fax: + 49 9131 85 28311.
E-mail address: cordt.zollfrank@ww.uni-erlangen.de (C. Zollfrank).
this trend may be considered dramatic and is seen in the USA, Japan and
particularly in Europe [12]. Unquestionably, barrier precautions like
meticulous hygienic measures, hand washing remain the main goal
in the prevention of NI. However, it has been described that the surfaces
of the inanimate environment such as instruments, cables, switches,
accessories, doorknobs, bed gear, blankets and sanitary installations
can act as a reservoir for multi-resistant pathogens and in combination
with invasive devices that bypass the body's natural lines of defence
provide an easy route for infection [13,14]. Therefore, there is increased
evidence for the occurrence of NI connected to the transmission from
contaminated surfaces [15–17], indicating that the concept of prevention cannot be limited to hand washing or antimicrobial biomaterials.
Germ free surfaces close to patients in sensitive areas assume highest
priority. Therefore, effective strategies to reduce the number of NI
by infection transmission through genuine bacteria free inanimate surfaces will increase the state of health in society. Current approaches to
decrease microbial contamination on inanimate surfaces are either preventive or biocidal. The first category aims at preventing adhesion of
the infectious agents on the surface through an anti-adhesive coating.
These include poly(ethylene glycol) [18], diamond-like carbon [19],
self-cleaning surfaces (Lotus effect) [20,21], and amphiphilic polymer
coatings [22,23]. Since the infectious agents are not eliminated, their
presence might be still a high risk for vulnerable patients. A more
reliable approach is the use of biocidal coatings on materials surfaces
[24]. Successfully applied technologies employ disinfectants such as
Triclosan [25] or inorganic antimicrobials such as silver ions [26,27],
0928-4931/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.msec.2011.09.010
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copper ions [28] and photocatalytic agents (e.g. TiO2) [29,30]. Apart
for photocatalytically active materials, the techniques make use of
the diffusion properties of the respective antimicrobial from the surface
to the attached microbes, hereby decreasing their viability rate. Existing
antimicrobial modified surfaces suffer from a number of limitations,
including the rapid release of the adsorbed antibiotic after implantation [24]. This results in a time limited antimicrobial activity [31]. An
enhanced frequency of resistance of the infectious agents has to be encountered by increasing exposure. This is eminently true for antibiotics
and some disinfectants where cross-resistance with antibiotics has
been described. Microorganisms also developed resistance even to inorganic antimicrobials. For many existing antimicrobials e.g. copper and
silver ions cytotoxicity has been reported on mammalian cells [32],
which limits their application in biomedical devices and healthcare
environments. Last but not least, these materials often lack cost efficiency.
It seems therefore essential to develop a new materials concept to cope
with problems encountered with current technologies.
A cost-efficient method for the production of coatings with an
antimicrobial effect might be the sol–gel technique. In a sol, small
particles of a solid are dispersed in a liquid. Thereby, the precursors
are metallic or metalloid elements, which are surrounded by several
ligands. By hydrolysis and a condensation step, a three-dimensional
network is formed. Thereby, the viscosity is changed and the sol is transformed to a gel and finally to a solid, which normally is an oxide ceramic
[33]. The sol–gel technique is an often used method for the production of
thin layers of different materials for several applications, such as optic
[34], electrochromic [35], photocatalytic [36] and antibacterial [37–39]
use. Thereby, the antibacterial effect of standard systems strongly correlates to silver particles dispersed in the coating. Upon contact with
water, the silver will be dissolved by the formation of ions, which
are responsible for the reduction of the bacteria concentration. Thus,
the antibacterial effect is time limited and strongly correlates to the silver
concentration.
Herein, the authors report on the development of an innovative
approach for an antimicrobial coating using the antibacterial effect
of transition metal acids. Molybdenum oxides (MoOx, 2 b x b 3) and
tungsten oxides (WOx, 2 b x b 3) in contact to water are exceptionally
effective agents against nosocomial pathogens such as Staphyloccocus
aureus and Pseudomonas aeruginosa. Although molybdenum and tungsten oxides are renowned for their gas sensing, [40] and electrochromic
characteristics [35] in engineering applications, the anti microbial properties of these materials have not been investigated so far. Recently, the
antimicrobial properties of molybdenum and tungsten oxides were
shown for a number of substrates including polymers, metals, glasses
and ceramics [41]. The investigated approach is a facile and cheap way
to produce long-lasting antibacterial surfaces. It can be applied to most
standard materials and thus can be a major goal for the reduction of NI
in hospitals.
2. Materials and methods
2.1. Preparation of samples
In a typical experiment, the respective inorganic powders (Mo:
3.8 μm; MoO2: 3.6 μm, and MoO3: 15.9 μm) were mixed with an acrylic
resin powder (Transoptic, Buehler) attaining concentrations up to 50
wt.-%. These high concentrations of the transitions metal acids were
selected at first to ensure a sufficient amount of the active inorganic
particles displayed on the surface of the samples. The powder mixtures
were pressed into small rods (2 cm in length, 1 cm in diameter) and
subsequently cured.
The PU tubes were manufactured by extrusion of a PU granulate.
Various PU tubes containing different amounts of MoO3 filler particles
(d50 = 0.8 μm) were fabricated. The MoO3 filled PU was prepared by
mixing the PU granulate together with a given fraction of MoO3
particles (0.5, 2.0 and 5.0 wt.%). Unmodified and MoO3 filled PU
tubes were manufactured on an existing industrial machine line.
Ti rods from an electron melt process (Ti–6Al–4V) exhibiting a rough
surface with a diameter of 2 mm were dip-coated in Mo containing sol,
which was prepared by dissolution of metallic Mo in a mixture of acetic
acid and hydrogen peroxide solution. The Mo oxide based gel coated Ti
rods were air-dried and annealed at 300, 500 and 700 °C to assess the
influence of the oxide phase formation during annealing of the Mo
oxide coating.
2.2. Investigated microorganisms
The antimicrobial activity of molybdenum oxide has been investigated with respect to S. aureus ATCC 25923 (MRSA) and P. aeruginosa
ATCC 10145.
2.3. Investigation of antimicrobial activity
The microorganisms are stored on slant agar at −25 °C single colonies
have been grown on Columbia agar with the addition of 5% defibrinated
sheep blood for 12 hours at 37 °C. The inoculum count was estimated
according to the Mc Farland standard [42]. In addition the concentration
was evaluated by a photometric method: an OD of 0.8 at 475 nm reveals
a concentration of 109 CFU/ml of Staphylococcus aureus. Final concentrations were determined with several 1:10 dilutions until colonies were
countable. A dispersion of 10 9 colony forming units (CFU)/ml in
1/4 N saline was prepared by harvesting bacteria of an overnight culture. The samples rods are incubated with a solution containing the
109 CFU/ml for 4 h. This time is sufficient that bacteria are colonising
the surface of the sample and start biofilm formation. After incubation
the samples were subsequently rolled onto an agar plate (OXOID TSB
nutrient agar with the addition of 5% defibrinated sheep blood,
OXOID) to document colonisation and then transferred into sterile
1/4 N saline. This procedure was repeated every three hours for a total
of 12 hours. The agar plates were incubated at 37 °C for 24 h, Fig. 1a.
At the same time negative controls were included in each experiment.
By this method (“roll-on” culture method) not only the strength of
adherence, proliferation but also the bactericidal activity of microorganisms colonising a surface is assessed [27,43–45].
Additionally, the investigation of antimicrobial activity was documented by a semi quantitative method [46]. A microplate lid was
equipped with test probes forming a ‘comb’. The teeth of the ‘comb’
comprise the materials to be tested and fit precisely into the wells of
a commercial 96-well microplate. The teeth are colonised by turning
the lid upside down and filling it with a microbial dispersion. After
removal of the “comb”, the growth of released daughter cells into
the minimal medium was stimulated by addition of trypticase soy
broth. Proliferation of microbes was followed online by optical density
measurements at 578 nm using a kinetic detection mode. Plates were
read with a microplate reader (Spectramax 250, Molecular Devices,
Sunnyvale, California, USA). The processed data provided a time proliferation curve as testing result for each assay. If bacteria were partially or
completely inactivated by the sample surface they were able to seed
only few or even no cells resulting in lagging or absence of bacterial
growth [47]. The time needed for a population to proliferate to an optical density (OD) of 0.2 is defined as the onset time tOD. The difference
time tX of the onset time for the reference sample tOD,ref (no MoO3)
and the onset time for the modified samples tOD,X were calculated
with respect to the tOD,ref. An antimicrobial activity of the respective
samples is defined for difference times larger than 6.0 corresponding
to a threefold order of magnitude increase of onset of cell proliferation.
2.4. Cytotoxicity evaluation
Cytotoxicity was monitored using the 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay (MTT assay) [48]. This
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Fig. 1. a) Experimental protocol for the “roll-on” test to evaluate the antimicrobial activity of a materials surface. The sample rods were incubated with a bacteria dispersion and
rolled over a nutrient agar plate. The growth of transferred bacteria on the agar plates was evaluated. This procedure was repeated every three hours for 12 h. Tested agar plates
for S. aureus and P. aeruginosa for b) oxidised molybdenum, c) polymers modified with molybdenum metal, molybdenum dioxide MoO2 and molybdenum trioxide MoO3.
assay measures the reducing potential of the cell using a colorimetric
reaction and determines the metabolic activity of a cell. Viable cells will
reduce the MTT reagent to a coloured formazan product, which can be
measured by colorimetric techniques. This test system determines the
metabolic activity of test cells and impairment by toxic substances.
For the measurement, a 24 h eluate of a 10 cm2 surface in 10 ml
physiologic saline was prepared. Dilutions 1:1, 1:2, 1:5 and 1:10 of the
eluate with a dispersion of MRC5 mouse lung fibroblast cells were
used as control dispersions with 5 × 105 MRC5 cells in 1/4 N saline. The
number of test cells was adjusted to 5 × 105 cells in Dulbeccos modified
Eagle Medium. Simultaneously two control media were added: as negative control for non-cytotoxicity 5 × 105 MRC5 mouse fibroblasts in
1/4 N saline and as positive control for cytotoxicity MRC5 cells with
0.1% copper sulphate. To 0.5 ml of these supernatants 10 μl of farmazan
was given. The colour change was measured in a photometer as optical
density (OD) at a wavelength of 475 nm after 4 h incubation at 37 °C.
3. Results
3.1. Investigation of antimicrobial activity
The “roll-on” culture method was applied as a screening method for
the assessment of the antimicrobial activity of our modified sample
rods, Fig. 1. The results indicate, that pure surface oxidised Mo
metal already showed antimicrobial activity, Fig. 1b. Very little colonies
were visible at 3 h, after 6, 9 and 12 h virtually no microorganisms were
detected. In contrast, negative controls showed unaffected growth of
bacteria throughout the entire experiment. To further elucidate, which
of the Mo phases are the active component, various Mo and MoOx
(x= 2 or 3) loaded polymer samples were tested. Pure Mo did not
show any antimicrobial activity for P. aeruginosa. S. aureus showed
a decrease in the number of detectable colonies after 9 h, Fig. 1c. In
contrast, MoO2 loaded polymer sample exhibited a noticeable antimicrobial activity. After 6 h most of the infectious agents were eradicated
from the incubated sample surface. The surface of the sample was free of
bacteria after 9 h. The antimicrobial activity drastically increased by the
addition of MoO3 particles to the polymer matrix. No infectious agents
could be transferred after 3 h in the roll-on test. This means, that the bacteria vanished or were inactivated by the MoO3 phase displayed on the
surface of the polymer samples. This astonishing new effect of antimicrobial activity for transition metal acids gives rise for a completely new
materials surface design to prevent transmission of infectious agents
and thus to decrease the risk of NI.
3.2. Effect of MoO3 concentration
The potential of our materials concept using antimicrobial active
MoO3 particles was further evaluated on polyurethane (PU) hollow
tubes, which are used as an isolation of electric cables. The outer surface of the PU tubes was subjected to the “roll-on” test to evaluate the
antimicrobial activity, Fig. 2. Although, the entire polymer composite
was filled with inorganic particles, part of the MoO3 filler particles
were displayed on the surface of the PU tubes. A scanning electron
microscopy (SEM) micrograph of an unfilled PU tube is shown in
Fig. 2a. The pure PU surface did not show any antimicrobial activity.
Colonies of S. aureus and P. aeruginosa were transferred from the incubated tubes even after 12 h. The MoO3 particles of the modified PU
tubes are clearly detectable in SEM investigation (back-scattered electron
mode, BSE) as white spots, Fig. 2b–d. Whereas the particles were hardly
visible at a filler content of 0.5 wt.%, the MoO3 agglomeration could
be undoubtedly identified at filler concentrations of 2.0 and 5.0 wt.%.
The inset in Fig. 2c shows a magnification of the agglomerate in range
of a few microns consisting of submicron-sized MoO3 particles. Evaluation of the “roll-on” test distinctly showed a considerable antimicrobial
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Fig. 2. SEM micrographs (BSE mode) of the surfaces of extruded unmodified and MoO3 filled PU tubes and corresponding tested agar plates for S. aureus and P. aeruginosa: a) unmodified
PU tube, b) 0.5 wt.% MoO3 (white circles highlight selected MoO3 particles), b) 2.0 wt.% MoO3 and c) 5.0 wt.% MoO3.
activity for MoO3 filler concentration of 0.5 wt.%, Fig. 2b. A substantial
amount of bacteria was still viable after 6 h on the MoO3 modified PU
tube, so that colonies of S. aureus and P. aeruginosa could be detected
on the agar plates. After 12 h colony density due to bacteria transfer
was decreased. This means that the MoO3 exhibits an antimicrobial
activity, but the modified PU tubes were not free of bacteria. Increasing
the MoO3 fraction resulted in an increase of the microbial activity. Significant reduction of the bacteria on the PU surfaces was obtained for
2.0 wt.% MoO3. Some transferred S. aureus were still present after 6 h
whereas P. aeruginosa was nearly absent. At 5.0 wt.% MoO3 filler concentration, the modified PU rods were virtually free of bacteria after
9 h. Both S. aureus and P. aeruginosa were hardly detectable after the
“roll-on” test on the agar plates. A SEM investigation of S. aureus on
unmodified surface of the PU tubes showed, that the cells are intact
and do not show any degradation effects, Fig. 3a. Contrary, S. aureus on
MoO3 filled PU tubes (5.0 wt.%) exhibited severe deterioration after
60 min of the cells, which resulted in cell death, and thus inactivation of
the infectious agent, Fig. 3b. A similar SEM observation could be obtained
on E. coli. The bacteria are fully intact on unmodified PU tube surfaces,
Fig. 3c, whereas degradation of the cells was clearly visible on 5.0 wt.%
MoO3 modified PU tubes after 10 min, Fig. 3d. As a result it could be distinctly shown, that MoO3 modified PU tubes, even at low concentration
of MoO3 (0.5 wt.%) exhibit a distinct highly efficient antimicrobial activity. Increasing the filler concentration to 5.0 wt.% resulted in an enhanced
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Fig. 3. a) Individual cell of S. aureus on an unmodified surface of a PU tube (60 min), b) deteriorated cell of S. aureus on PU tube containing 5.0 wt.-MoO3 60 min after incubation,
c) intact E. coli. on an unmodified surface of a PU tube (10 min), d) deteriorated cells of E. coli. on a PU tube containing 5.0 wt.-MoO310 min after incubation.
antimicrobial activity of the modified PU tubes, which was sufficient to
remove almost all bacteria after 9 h contact on the tube surface.
The semi-quantitative method for the investigation of antimicrobial
activity showed typical time-proliferation curves of microorganisms for
the microplate proliferation assay of our samples, Fig. 4a. The unmodified samples were measured as a reference and the corresponding
curve shows uninhibited growth. The curves of the MoO3 modified
samples were shifted indicating a delayed proliferation. The calculated
difference time t0.5 from the diagram for the 0.5 wt.% MoO3 modified
was 13.5, and the calculated difference time for t2.0 and t5.0 were 17.5
and 33.0, respectively. Since these values are well above the threshold
value of 6.0, the highly efficient antimicrobial activity of the MoO3
modified samples was independently confirmed.
3.3. Cytotoxicity measurements
The high measured OD of the negative references and our modified
MoO3 samples indicates, that the MoO3 modified samples are noncytotoxic, in contrast to the copper containing positive samples, where
the low observed OD values points to the expected high cytotoxicity,
Fig. 4b.
3.4. Temperature treatment
Further evidence of the antimicrobial activity of MoO3 and the
active phases was obtained on modified titanium (Ti) rods, Fig. 5. The
Mo oxide based yellow gel coating is clearly visible on the as-prepared
dip-coated Ti rods, Fig. 5c and d. According to X-ray diffraction, the nearly
amorphous gel coating consisted of a mixture of molybdates containing
[Mo3O10]2− ions and hydrated MoO3, Fig. S1. After annealing the gel
layer was transformed into slightly blue crystalline MoO3 with broad
peaks consisting of a mixture of monoclinic and orthorhombic crystal
structures. The appearance of the coating did not change. The antimicrobial properties were assessed using the “roll-on” test. The Mo
oxide gel coating on the Ti rods treated at 300 °C exhibited an excellent antimicrobial activity against P. aeruginosa. Transmission of the
bacteria was no longer observed after 3 hours indicating a bacteria
free materials surface, Fig. 3e. The annealing treatment resulted in
further crystallisation of the gel product. The fraction of MoO3 orthorhombic phase was increased. However, a significant decrease of the
antimicrobial activity was observed for samples treated at 500 °C.
There was a considerable transmission of bacteria even after 12 h.
A distinct antimicrobial activity could no longer be observed for the
samples treated at 700 °C. The samples were fully infectious after
12 h. After annealing at 700 °C, the nearly white MoO3 was well crystallised and consisted almost exclusively of the orthorhombic crystal
phase. For reason of comparison, a silver containing Ti-alloy, which
was expected to show an enhanced antimicrobial activity was also
tested, Fig. 5f. The results indicated extensive transmission of bacteria
after 12 h. As a result, Ti metal alloy, even if it contains silver, does not
show a noticeable antimicrobial activity. On the other hand, it could
be definitely shown, that the sol–gel derived Mo oxide coating on Ti
alloy exhibits an extraordinary effective antimicrobial activity after
annealing at 300 °C. By further annealing and transformation into crystalline Mo oxide phases, the antimicrobial activity is lost. The crystal
structure development with temperature indicates, that the monoclinic phase might be correlated with the antimicrobial activity. It
could be, however, shown for the first time, that sol–gel derived Mo
oxide coatings are efficient coatings to prevent growth of bacteria on inanimate surfaces.
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[49–52]. Thus, this mechanism is fairly non-specific (active against
a broad spectrum of gram-positive and gram-negative bacteria) and,
in contrast to antibiotics, does not produce bacteria resistant to this
mode of action. The acidic surface inhibits in many cases proliferation
of the cells and the formation of biofilms leading to elimination of the
infectious agents within six to nine hours. The acid activity refers to
the diffusion of H3O+ ions through the cell membranes. This results in
a distortion of the sensitive pH-equilibrium as well as the enzyme and
transport systems of the cell [53]. There is also a disruption of the
DNA helix [54]. Furthermore, energy is required from the cell to adjust
the distorted pH-equilibrium resulting in further weakening of the cell
[55]. Microorganisms growing in an acidic environment experience
also an efficient blockage of adherence and biofilm formation. However,
the direct contact of the microorganism with the acidic materials surface appears to be important.
As described above, it is necessary for the antibacterial effect that the
material is in contact with water. Despite this, leaching and dissolution
of MoO3 is not relevant in our approach since we are dealing with
inanimate materials surface that is not exposed to a permanent aqueous
environment. The only contact with water might be through touching
the surfaces (e.g. healthcare staff, patients) as well as general humidity.
Under such “dry” static conditions leaching or dissolution of the MoO3
from the materials surface will only occur to a small amount, if any at
all, and will only be of minor relevance. Additionally, it is well known,
that MoO3 exhibits water-solubility only at high pH-values. The solubility of MoO3 in water at neutral pH is 56.0 ±0.1 mg/l [56], and thus
very low. Interestingly, an antimicrobial activity of a MoO3 particle
dispersion in water could not be confirmed [57]. It was shown, that
MoO3 up to a concentration of 1 g/l had no effect on cell counts of
Acinetobacter sp. and several other bacteria. These results indicate
the low cytotoxicity and the importance of a direct contact between
the bacterium and the material surface.
5. Conclusions
Fig. 4. a) Evaluation of the antimicrobial activity using a semi quantitative microplate
assay, [48] b) assessment of the cytotoxicity of the MoO3 modified samples.
4. Discussion
The antimicrobial principle of transition metal oxides (MoO3) can
be related to an acidic surface reaction (release of hydroxonium ions)
according to the following reactions (1) and (2):
MoO3 þ H2 O⇄H2 MoO4
ð1Þ
þ
2−
H2 MoO4 þ 2H2 O⇄2H3 O þ MoO4
ð2Þ
First molybdic acid in the hydrated form of molybdenum trioxide
is formed on the surface of the modified materials, reaction (1). The
simplest solid form is the monohydrate MoO3 × H2O (H2MoO4), but
the dihydrate MoO3 ×2H2O is also known. Hydroxonium ions (H3O+)
will be released from H2MoO4 in the presence of water forming the corresponding molybdates (MoO42 −). In the equilibrium state, the molybdates will be retransformed into molybdic acid H2MoO4.
Acidic surfaces are well known to generally slow down bacterial and
fungal growth and effectively kill microorganisms at pH values of 3.5–
4.0 (e.g. staphylococci, streptococci, enterococci, Legionella pneumophila,
Lactobacillus acidophilus spp., Candida spp., Aspergillus spp.). Many gramnegative microorganisms are killed at even higher pH values up to
5.5 (e.g. E. coli, Pseudomonas aeruginosa, Clostridia, Campylobacter)
We presented a novel materials concept to permanently prevent
growth of infectious agents (bacteria) on various materials surfaces,
which were modified with transition metal oxides that can release
hydronium ions in contact with water. We confirmed, that MoO3
exhibits a highly efficient antimicrobial activity towards severe infectious
agents such as S. aureus and P. aeruginosa. Polymers (PU) and metal (Ti)
surfaces modified with MoO3 particles or sol–gel based coatings were virtually free of bacteria within 6 h after incubation with an infectious solution. We could show, that the MoO3 modified samples are not cytotoxic.
The antimicrobial activity of transition metal acid MoO3 is related to
their surface acidity involving the intermediate formation of molybdic
acid. Our developed materials concept is applicable to almost any materials surface, because the antimicrobial active agents can be introduced
using conventional processing extrusion or in coatings. Materials with
an antimicrobial coating are important to decrease the transfer and
spreading of infectious agents wherever public interaction is involved.
The modification of materials surfaces with antimicrobial MoO3 might
be therefore important for materials used in public transportation and
other frequented locations. The decrease of nosocomial pathogens such
as S. aureus and P. aeruginosa is extremely relevant in healthcare environments, where transfer of infectious agents can cause severe infections of
already weakened patients. Our developed MoO3 based antimicrobial
materials concept is of general importance to decrease the risk of
health care associated infections, which is one of the most important
causes of disease at present. Therefore, the modification of materials
surfaces with antimicrobial active transition metal acid is also of
general interest to decrease transmission of infectious agents in public
environments.
Supplementary materials related to this article can be found online
at doi:10.1016/j.msec.2011.09.010.
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Fig. 5. SEM micrographs of the a and b) Ti rods and c and d) MoO3 gel coated Ti rods, e) antimicrobial activity of the MoO3 gel coated Ti rods after annealing at 300, 500 and 700 °C,
f) antimicrobial evaluation of a reference titanium alloy sample.
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Acknowledgment
The authors gratefully acknowledge financial support from the
German Research Foundation (DFG) under contract ZO113/13-1.
References
[1] L.A. Herwaldt, J.J. Cullen, D. Scholz, P. French, M.B. Zimmerman, M.A. Pfaller, R.P.
Wenzel, T.M. Perl, Infect. Control Hosp. Epidemiol. 27 (2006) 1291.
[2] K. Page, M. Wilson, I.P. Parkin, J. Mater. Chem. 19 (2009) 3819.
[3] D.J. Ecker, K.C. Carroll, ASM News 71 (2005) 576.
[4] D.L. Paterson, Clin. Infect. Dis. 43 (2006) S41.
[5] D.L. Paterson, Am. J. Med. 119 (2006) S20.
[6] A. Legras, D. Malvy, A.I. Quinioux, Intensive Care Med. 24 (1998) 1040.
[7] I.I. Raad, H.A. Hanna, M. Boktour, N. Jabbour, R.Y. Hachem, R.O. Darouiche, Infect.
Control Hosp. Epidemiol. 26 (2005) 658.
[8] E.M. D'Agata, Infect. Control Hosp. Epidemiol. 25 (2004) 842.
[9] J.I. Tokars, C. Richards, M. Andrus, Clin. Infect. Dis. 39 (2004) 1347.
[10] R. Gaynes, J.R. Edwards, Clin. Infect. Dis. 41 (2005) 848.
[11] S.R. Norrby, C.E. Nord, R. Finch, Lancet Infect. Dis. 5 (2005) 115.
[12] M.R. Mulvey, A.E. Simor, Can. Med. Assoc. J. 180 (2009) 408.
[13] S.J. Dancer, J. Hosp. Infect. 56 (2004) 10.
[14] D.G. Maki, W.A. Agger, Medicine 67 (1988) 248.
[15] A. Bhalla, N.J. Pultz, D.M. Gries, A.J. Ray, E.C. Eckstein, D.C. Aron, C.J. Donskey, Infect.
Control Hosp. Epidemiol. 25 (2004) 164.
[16] J.M. Boyce, G. Potter-Bynoe, C. Chenevert, T. King, Infect. Control Hosp. Epidemiol.
18 (1997) 622.
[17] D. Talon, J. Hosp. Infect. 43 (1999) 13.
[18] R.G. Chapman, E. Ostuni, M.N. Liang, G. Meluleni, E. Kim, L. Yan, G. Pier, H.S. Warren,
G.M. Whitesides, Langmuir 17 (2001) 1225.
[19] R.A. Hauert, Diamond Relat. Mater. 12 (2003) 583.
[20] W. Barthlott, C. Neinhuis, Planta 202 (1997) 1.
[21] A. Okada, T. Nikaido, M. Ikeda, K. Okada, J. Yamauchi, R.M. Foxton, H. Sawada, J.
Tagami, K. Matin, Dent. Mater. J. 27 (2008) 256.
[22] S.F. Rose, S. Okere, G.W. Hanlon, A.W. Lloyd, A.L. Lewis, J. Mater. Sci. Mater. Med.
16 (2005) 1003.
[23] G. Cheng, Z. Zhang, S.F. Chen, J.D. Bryers, S.Y. Jiang, Biomaterials 28 (2007) 4192.
[24] A.M. Klibanov, J. Mater. Chem. 17 (2007) 2479.
[25] R.O. Darouiche, M.D. Mansouri, P.V. Gawande, S. Madhyastha, J. Antimicrob.
Chemother. 64 (2009) 88.
[26] S. Silver, FEMS Microbiol. Rev. 27 (2003) 341.
[27] U. Samuel, J.P. Guggenbichler, Int. J. Antimicrob. Agents 23 (2004) S75.
[28] M.J. Domek, M.W. LeChevallier, S.C. Cameron, G.A. McFeters, Appl. Environ.
Microbiol. 48 (1984) 289.
[29] Z. Huang, P.C. Maness, D.M. Blake, E.J. Wolfrum, S.L. Smolinski, W.A.J. Jacoby,
Photochem. Photobiol. A Chem. 130 (2000) 163.
[30] R. Asahi, T. Morikawa, T. Ohwaki, K. Aoki, Y. Taga, Science 293 (2001) 269.
[31] G. Donelli, I. Fancolini, J. Chemother. 13 (2001) 595.
[32] B.S. Atiyeh, M. Costagliola, S.N. Hayek, S.A. Dibo, Burns 33 (2007) 139.
[33] C.J. Brinker, G.W. Scherrer, Sol–gel-Science, Academic Press Inc, San Diego, 1990
2.
[34] H. Bach, H. Schroeder, Thin Solid Films 1 (1967) 255.
[35] C.S. Hsu, C.C. Chan, H.T. Huang, C.H. Peng, W.C. Hsu, Thin Solid Films 516 (2008)
4839.
[36] K. Gutbrod, P. Greil, C. Zollfrank, Appl. Catal. B Environ. 103 (2011) 240.
[37] H. Hradecká, J. Matoušek, Ceramics-Silikáty 54 (2010) 219.
[38] O. Akhavan, E. Ghaderi, Surf. Coat. Technol. 204 (2010) 3676.
[39] P. Tatar, N. Kiraz, M. Asiltürk, F. Sayilkan, H. Sayilkan, E. Arpaç, J. Inorg. Organomet.
Polym. Mater. 17 (2007) 525.
[40] K. Galatsis, Y.X. Li, W. Wlodarski, K. Kalantar-zadeh, Sens. Actuators B 77 (2001)
478.
[41] J.P. Guggenbichler, N. Eberhardt, H.P. Martinez, H. Wildner, PCT/EP2007/009814
(2007).
[42] J. McFarland, J. Am. Med. Assoc. 14 (1907) 1176.
[43] J.P. Guggenbichler, Materialwiss. Werkstofftech. 34 (2003) 1145.
[44] J.P. Guggenbichler, Antibiot. Monit. 20 (2004) 52.
[45] J.P. Guggenbichler, Biomaterialien 5 (2004) 228.
[46] T. Bechert, P. Steinrücke, J.P. Guggenbichler, Nat. Med. 6 (2000) 1053.
[47] V. Alt, T. Bechert, P. Steinrucke, M. Wagener, P. Seidel, E. Dingeldein, E. Domann,
R. Schnettler, Biomaterials 25 (2004) 4383.
[48] T. Mosmann, J. Immunol. Methods 65 (1983) 55.
[49] J.M. Goepfert, R. Hicks, J. Bacteriol. 97 (1969) 956.
[50] G. Bergson, H. Arfinnsson, O. Steingrimmson, H. Thomar, Antimicrob. Agents
Chemother. 45 (2010) 3209.
[51] J.J. Kabara, W.J. McKillip, E.A. Sedor, I. Aminimidesi, J. Am. Oil Chem. Soc. 52
(1975) 316.
[52] E.J. Foley, F. Herrmann, S.W. Lee, J. Invest. Dermatol. 53 (1947) 422.
[53] A. Plumridge, S.J.A. Hesse, A.J. Watson, K.C. Lowe, M. Stratford, D.B. Archer, Appl.
Environ. Microbiol. 70 (2004) 3506.
[54] C. Schüller, Y.M. Mamnun, M. Mollapour, G. Krapf, M. Schuster, B.E. Bauer, P.W.
Piper, K. Kuchler, Mol. Biol. Cell 15 (2004) 706.
[55] H.J. Ra, W.C. Parks, Matrix Biol. 26 (2007) 587.
[56] P. Venkataramana, V.R. Krishnan Radiochem, Radioanal. Lett. 53 (1982) 189.
[57] S.L. Percival, J. Ind. Microbiol. Biotechnol. 23 (1999) 112.
Please cite this article as: C. Zollfrank, et al., Materials Science and Engineering C (2011), doi:10.1016/j.msec.2011.09.010