Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
[0001 ] Synergistic Antimicrobial Compositions and Methods of Inhibiting
Biofilm
Formation
FIELD OF THE INVENTION
[0002] This invention relates to synergistic antimierobial compositions which
inhibit biofilm
formation on or in medical devices such as catheters as well as other devices.
BACKGROUND OF THE INVENTION
(0003] Biofilms are medically and industrially important because they can
accumulate on a wide
variety of substrates and are resistant to antimicrobial agents and
detergents. Microbial biofilms develop
when microorganisms irreversibly adhere #o a surface and produce extracellular
polymers that facilitate
adhesion and provide a structural matrix. Therefore inhibiting adhesion to
surfaces is important. This
surface may be inert, non-living material or living tissue.
[0004] Biofilm-associated microorganisms behave differently from planktonic
(freely suspended)
organisms with respect to growth rates and ability to resist antimicrobial
treatments and therefore pose a
public health problem. Many chronic infections that are difficult or
impossible to eliminate with
conventional antibiotic therapies are known to involve biofilms. A partial
list of the infections that
involve biofilms includes: otitis media, prostatitis, vascular endocarditis,
cystic fibrosis pneumonia,
meliodosis, necrotising faciitis, osteomyelitis, peridontitis, biliary tract
infection, struvite kidney stone
and host of nosocomial infections (Costerton, J.W., et al., Science, 284:1318-
1322, 1999).
(0005] Biofilms on indwelling medical devices may be composed of gram-positive
or gram-negative
bacteria or yeasts. Bacteria commonly isolated from these devices include the
gram-positive
Enterococcus faecalis (E, faecalis), Staphylococcus epidermidis (S
epidermidis), Staphylococcus aureus
CA 02452032 2004-08-04
(S. aureus), Streptococcus viridans (St. viridans); and the gram-negative
Escherichia coli (E. coli),
Klebsiella pneurnoniae (K. pneumoniae), Proteus mirabilis (P. mirabilis) and
Pseudomonas aeruginosa
(P. aeruginosa)(Donlan, R.M., Emerging Infectious Diseases, 7:277-281, 2001).
The organisms most
commonly isolated from urinary catheter biofilms are Staphylococcus
epidermidis, Enterococcus
faecalis, E. coli, Proteus mirabilis, Pseudomonas aeruginosa and Klebsielda
pneumoniae. Urinary
catheters and central venous catheters are notorious examples of infection
prone devices. Catheter-
associated urinary tract infection is the most common nosocomial infection.
Each year, more than 1
million patients in US hospitals acquire such an infection. Catheter-
associated urinary tract infection is
the second most common cause of nosocomial infections (Maki, D.G. and P.A.
Tambyah., Emerging
Infectious Diseases, 7:1-6, 2001).
[0006] In recent years, there have been numerous efforts to sequester
antimicrobials and antibiotics
on the surface of or within devices that are then placed in the vasculature or
urinary tract as a means of
reducing the incidence of device-related infections. These antimicrobial
agents are of varying chemical
composition and can include chelating agents (EDTA, EGTA, DTPA, etc.),
cationic polypeptides
(protamine, polylysine, lysozyme, etc.), surfactants (SDS, Tween-80~surfactin,
etc.), quaternary
ammonium compounds (benzalkonium chloride, tridodecyl methyl ammonium
chloride, didecyl
dimethyl ammonium chloride, etc.). The iron-sequestering glycoproteins such as
lactoferrin from milk
and ovotransfernn (conalbumin) from egg white are iron-binding glycoproteins,
which inhibit the
growth of certain bacteria by making iron unavailable for bacterial metabolism
(Bezkorovainy, A., Adv.
Exp. Med. Biol. 135:139-154, 1981).
[0007] The main methods of antimicrobial catheter preparation include
immersion or flushing,
coating, drug-polymer conjugate and impregnating (Tunny, M.M., et al., Rev.
Med. Microbiol., 74: 195-
205, 1996). In a clinical setting, suitable catheters can be treated by
immersion immediately prior to
placement, which offers flexibility and control to clinicians in certain
situations. Several studies have
examined the clinical efficacy of catheters coated with antimicrobial agents.
Polyurethane catheters
coated with minocycline and EDTA showed potential in reducing recurrent
vascular catheter-related
infections (Raad, I. I. et al., Clinical Infectious Diseases, 25: 149-151,
1997). Minocycline and rifampin
coatings have been shown to significantly reduce the risk of catheter-
associated infections (Raad, LI. et
al., Crit. Care Med., 26: 219-224, 1998). Minocycline coated onto urethral
catheters has been shown to
2
CA 02452032 2005-04-05
provide some protection against colonization (Darouiche, R.O., et al., Int. J.
Antimicrob. Ag. 8: 24-3-
247,1997). Johnson, et al., described substantial in vitro mtimicrobial
activity of a commercially
available nitrofurazone coated silicone catheter (Johnson, J.R., et al.,
Antimicrob. Agents.
Chemother. 43: 2,990-2,995,1999). The antibacterial activity of silver-
containing compounds as
antimicrobial coatings for medical devices has been widely investigated.
Silver-sulfadiazine used in
combination with chlorhexidine has received particular interest as a central
venous catheter coating
(Stickler, D.J., Curr. Opin. Infect. Dis.,,13:389-393,2000; Darouiche, R.O.,
et al., New Eng. J. Med.,
340: 1-8,1999). The loading of antimicrobial agents into medical devices by
immersion or coating
technologies has the advantage of being.relatively simple. However, the
limited mass of drug that
can be incorporated may be insufficient for a prolonged atitimicrobial effect,
and the release of the
drug following clinical insertion of the device is rapid and relatively
uncontrolled. A means of
reducing these problems is by direct incorporation of the antimicrobial agent
into the polymeric
. y
matrix of the medical device at the polymer synthesis stage or at the device
manufacture stage.
Rifampicin has been incorporated into silicone in an attempt to prevent
infection of cerebrospinal
fluid shunts with some success (Schierholz, J.M., et al., Biomaterials, 18:
839-844,1997), and
hexetidine in PVC was observed to decrease bacterial colonization (Jones,
D.S., et al., Pharm. Res.,
19: 818-824,2002). Iodine has also been incorporated into medical device
biomaterials. Coronary
stems have been modified to have antithrombogenic and antibacterial activity
by covalent attachment
of heparin to silicone with subsequent entrapment of antibiotics in cross-
linked collagen bound to the
heparinised surface (Fallgren, C., et al., Zent.Fur Bakt.-Int. J. Med. Micro.
Vir.; Paraotol. Infect.
Dis., 287:19-31,1998).
[0008) Charter, E.A., et al., disclosed a composition consisting of avidin,
ovotransferrin,
chicken immunoglobulins, chitosan, polylysine; protamine, nisin, EDTA,
rosemary, cinnemaldehyde,
allicin and eugenol for inhibiting the growth of microoragmisms on fruit,
vegetable, turfgrass and
other plant systems by the application of specific enzymes either alone or in
combination with
fungicidally active agents (US Pat. Application No. 20020001582, 2002). In US
Patent No.
6,187,768 (2001), Welle, C.J., et al., disclosed the method of preparing a kit
for flushing a medical
device. The kit includes a solution containing an antibiotic;, an
anticoagulant (protamine sulfate) and
an antithrombotic agent or chelating agent useful for preventing infections
caused by bacterial
growth in catheters. Budny, J.A, et al., discloses various antimicrobial
agents for anchoring to
biofilms (US Patent Application No. 20020037260, 2002). Raad, et al., in US
Patent No. 5,362,754
(1994) disclosed that pharmaceutical
CA 02452032 2003-12-04
compositions of a mixture of minocycline and EDTA were useful in maintaining
the patency of a
catheter port. Recently, Raad, L and R. Sheretz in US Patent No. 5,688,516
(1997) further disclosed that
effective catheter flush solutions could be prepared with non-glycopeptide
antimicrobial agent, an
antithrombic agent, an anticoagulant and a chelating agent selected from the
group consisting of EDTA,
EGTA and DTPA. US patent no. 6,187,768 to Welle et al. teaches the use of
several anticoagulants for
use in medical devices, including protamine sulfate. US patent no. 6,174,537
to Khan teaches the
flushing of intravascular catheters using EDTA in combination v~ith salts of
sodium, calcium and lactic
acid.
[0009j The methods currently in use for prevention of biofilms act at the
level of removal versus
formation of the biofilms. These methods are costly, often involve the use of
caustic chemicals, and
often provide only short-term prevention. In medical devices, various
techniques have been described
that incorporate potentially toxic metal ions in the form of metal salts into
materials that make up the
medical devices. The protection against biofilm formation lasts only as long
as the coating remains on
the device. A method of long-term prevention from biofilm formation that acts
at the level of prevention
of biofilm formation is needed. Also needed i~ a composition that allows for
low quantities of the
composition to be used effectively, thus reducing toxicity or other side
effects to the user or patient
without sacrificing effectiveness against biofilm formation. There is also a
need for antimicrobial
compositions that are environmentally friendly, medically acceptable, highly
effective and relatively
economical to manufacture on a commercial scale for preventing biofilm
formation in biomedical
devices.
SUMMARY OF THE INVENTION
[0010] Accordingly, one aspect of the invention provides a composition
comprising: (a) a small
amount of at least one iron-sequestering glycoprotein; and (b) a sparing
amount of at least one cationic
polypeptide, wherein the amount of each components (a) and (b) is sufficient
to form, in combination, a
synergistic, antimicrobial composition. In yet another alternative embodiment,
a composition of the
invention comprises: (a) a small amount of at least one iron-sequestering
glycoprotein and (b) a sparing
amount of at least one chelating agent, wherein the amount of each of
components (a) and (b) is
4
CA 02452032 2003-12-04
sufficient to form, in combination, a synergistic antimicrobial composition.
In yet another alternative
embodiment, a composition of the invention comprises: (a) a small amount of at
least one iron-
sequestering glycoprotein, (b) a sparing amount of at least one cationic
polypeptide and (c) a sparing
amount of at least one chelating agent, wherein the amount of each of
components (a), (b) and (c) is
sufficient to form, in combination, a synergistic antimicrobial composition.
[0011] Thus, the invention provides a composition for inhibiting bacterial
biofilm on devices
comprising: an iron-sequestering glycoprotein; a cationic polypeptide; and a
chelating agent. The
composition is effective against biofilms produced by bacterial species
selected from the group
consisting of Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus
epidermidis: The
composition is effective against biofilms produced by gram-negative bacterial
species selected from the
group consisting of Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae
and Pseudomonas
aeruginosa. The composition is effective against biofilms produced by gram-
positive bacterial species
selected from the group consisting of Enterococcus faecalis and Staphylococcus
epidermidis.
[0012] The invention also provides a composition for inhibiting bacterial
biofilm on devices
comprising an iron-sequestering glycoprotein; and a cationic polypeptide. The
composition is effective
against biofilms produced by bacterial species selected from the group
consisting of Staphylococcus
epidermidis, Enterococcus faecalis, E. coli, Proteus mirabilis, Pseudomonas
aeruginosa, Kdebsiella
pneumoniae, Staphylococcus aureus, Streptococcus viridans, Kdebsiella oxytoca,
Staphylococcus
saprophyticus, Providentia stuartii and Serratia marcescens, Klebsiella
pneumoniae, Pseudomonas
aeruginosa and Staphylococcus epidermidis. The composition is particularly
effective against biofilms
produced by bacterial species selected from the group consisting of
Escherichia coli, Proteus mirabilis,
Klebsiella pneumoniae Pseudomonas aeruginosa and Staphylococcus epidermidis.
[0013] The invention further teaches a composition for inhibiting bacterial
biofilm on devices
comprising: an iron-sequestering glycoprotein; and a chelating agent. The
composition is effective
against biofilrns produced by bacterial species selected from the group
consisting of Staphylococcus
epidermidis, Enterococcus faecalis, E. coli, Proteus mirabilis, Pseudomonas
aeruginosa, Klebsiella
pneumoniae, Staphylococcus aureus, Streptococcus viYidans, Klebsiella oxytoca,
Staphylococcus
saprophyticus, Providentia stuartii and Serratia marcescens, Klebsiella
pneumoniae, Pseudomonas
CA 02452032 2004-08-04
aeruginosa and Staphylococcus epidermidis. The composition is particularly
effective against
biofilms produced by Staphylococcus epidermidis.
[0014) In an embodiment, the iron-sequestering glycoprotein is between about
125 mg/L and
about 2000 mg/L of the composition. The cationic polypeptide is between about
12.5 mg/L and
about 200 mg/L of the composition. The chelating agent is between about 12.5
mg/L and about 200
mg/L of the composition.
[0015] The iron-sequestering glycoprotein may be selected from the group
consisting of
ovotransfernn, lactofernn and serotransfernn. The cationic polypeptide may be
selected from the
group consisting of protamine sulfate, polylysine, defensin, lactoperoxidase
and lysozyme. The
chelating agent may be selected from the group consisting of
Ethylenediaminetetraacetate (EDTA),
Ethyleneglycolbis(aminoethlyl)tetraacetate (EGTA),
Diethylenetriaminepentaacetic acid (DTPA),
Ethylenediaminedi(o-hydroxyphenylacetic) acid (EDDHA), Iminodiacetic acid
(IDA),
Cyclohexanediaminotetraacetic acid (CDTA),
Hydroxyethylethylenediaminetetraacetic acid
(HEDTA), Hydroxyethylnitrilodiacetic acid (HEIDA) and Nitrilotetraacetate
(NTA). In an
embodiment, the iron-sequestering agent is ovotransferrin, the cationic
polypeptide is protamine
sulfate, and the chelating agent is EDTA. The ovotransfernn may be present at
about 2 mglml, the
protamine sulfate may be present at about 0.2 mg/ml, and the EDTA may be
present at about 0.2
mg/ml.
[0016] The composition may further comprise one or more ingredients selected
from the
group consisting of: water, a binding or bonding or coupling agent, a
surfactant, a quaternary
ammonium compound, an antibiotic and a pH adjuster.
[0017) The invention also teaches methods of preparing a device comprising
treating at least
a surface of the device with the composition of the invention. The invention
also teaches methods of
preparing a device comprising coating a device with the composition of the
invention. The method
may further comprise treating the device with quaternary ammonium compound
before coating the
device with the composition. The quaternary ammonium compound may be selected
from the group
consisting of tridodecylmethyl ammonium chloride and benzalkonium chloride.
The composition
may further comprise hydrogel. The hydrogel maybe selected from the group
consisting of
polyvinylpyrrolidone-hydrogel, polyvinyl alcohol-hydrogel and polyethylene
glycol-hydrogel.
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(0018] The treated device may be a medical device. The device may be a
catheter. The catheter
may be an indwelling catheter. The indwelling catheter may be selected from a
group consisting of a
central venous catheter, a peripheral intravenous catheter, an arterial
catheter, a haemodialysis catheter,
an umbilical catheter, precutaneous nontunneled silicone catheter, a cuffed
tunneled central venous
catheter and a subcutaneous central venous port. The catheter may be selected
from a group consisting
of urinary catheter and a peritoneal catheter. The device may be selected from
the group consisting of
catheters, pacemakers, prosthetic heart valves, prosthetic joints, voice
prostheses, contact lenses, and
intrauterine devices. The device may be selected from the group consisting of
pipes, heat exchangers
and computer chips.
[0019] The invention also teaches methods of preparing a device comprising
incorporating the
composition of the invention into polymers which are used to form the device,
and methods of preparing
a device comprising impregnating the composition of the invention into the
device.
BRIEF DESCRTPTION OF THE DRAWINGS
[0020] Figure 1 is a bar graph showing the effects of ovotransferrin (OT),
protamine sulfate (PS) and
EDTA (ethylenediaminetetraacetate) alone and in combinations (OT+PS, OT+EDTA &
PS+EDTA) on
biofilm formation by E. coli.
[00211 Figure 2 is a bar graph showing the effects of ovotransferrin (OT),
protamine sulfate (PS) and
EDTA (ethylenediaminetetraacetate) alone and in combinations (OT+PS, OT+EDTA &
PS+EDTA) on
biofilm formation by Enterococcus faecalis. -
[0022] Figure 3 is a bar graph showing the effects of ovotransferrin (OT),
protamine sulfate (PS) and
EDTA (ethylenediaminetetraacetate) alone and in combinations (OT+PS, OT+EDTA &
PS+EDTA) on
biofilm formation by Klebsiella pneumoniae.
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[0023] Figure 4 is a bar graph showing the effects of ovotransferrin (OT),
protamine sulfate (PS) and
EDTA {ethylenediaminetetraacetate) alone and in combinations {OT+PS, OT+EDTA &
PS+EDTA) on
biofilm formation by Pseudomonas aeruginosa.
(0024] Figure 5 is a bar graph showing the effects of ovotransferrin (OT),
protamine sulfate (PS) and
EDTA (ethylenediaminetetraacetate) alone and in combinations (OT+PS, OT+EDTA &
PS+EDTA) on
biofilm formation by Proteus mirabilis.
[0025] Figure 6 is a bar graph showing the effects of ovotransfen-in (OT),
protamine sulfate (PS) and
EDTA (ethylenediaminetetraacetate) alone and in combinations (OT+PS, OT+EDTA &
PS+EDTA) on
biofilm. formation by Staphylococcus epidermidis.
[0026] Figure 7 is a bar graph showing the effects of ovotransfernn (OT),
protamine sulfate (PS) and,
EDTA alone and in combination (OT+PS+EDTA) on biofilm formation by E. coli.
[0027] Figure $ is a bar graph showing the effects of ovotransferrin (OT),
protamine sulfate (PS) and
EDTA alone and in combination (OT+PS+EDTA) on biofilm formation by
Enterococcus faecalis.
[0028] Figure 9 is a bar graph showing the effects of ovotransferrin (OT),
protamine sulfate (PS) and
EDTA alone and in combination (OT+PS+EDTA) on biofilm formation by Pseudomonas
aeruginosa.
[0029] Figure 10 is a bar graph showing the effects of ovotransfernn (OT),
protamine sulfate (PS)
and EDTA alone and in combination (OT+pS+EDTA) on biofilm formation by
Klebsiella pneumoniae.
[0030] Figure 11 is a bar graph showing the effects of ovotransferrin (OT),
protamine sulfate (PS)
and EDTA alone and in combination (OT+PS+EDTA) on biofilm formation by Proteus
mirabilis.
[0031] Figure 12 is a bar graph showing the effects of ovotransferrin (OT),
protamine sulfate (PS)
and EDTA alone and in combination (OT+pS+EDTA) on biofilm formation by
Staphylococcus
epidermidis.
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[0032] Figure 13 shows the effects of synergistic composition on biofilm
formation by Proteus
mirabilis, Pseudomonas aeruginosa and Staphylococcus epidermidis in a
catheter.
DETAILED DESCRIPTION OF THE INVENTION
[0033] The present inventor has found that an unexpectedly high level of
synergy occurs in
antimicrobial compositions that contain at least one iron-sequestering
glycoprotein, one cationic
polypeptide and one chelating agent. The synergy is evidenced by the small
quantities of each of these
compounds that need to be used to produce an effective antimicrobial
composition. The necessary
overall amount of the compounds is less than that which would be required if
any of the compounds
were to be used on their own. In particular, it is possible to use small
amounts of iron-sequestering
glycoproteins, which can be expensive but are biologically acceptable, with
small amounts of cationic
polypeptides, which are also biologically acceptable and small amounts of
chelating agents, which are
biologically acceptable at lower concentrations and are effective
antimicrobials. Synergy was found with
antimicrobial compositions that contain at least one iron-sequestering
glycoprotein with at least one
cationic polypeptide or at least one chelating agent.
[0034] The present invention teaches synergistic antimicrobial compositions
offering superior anti-
biofilm activity, containing combinations of iron-sequestering glycoproteins
with other antimicrobial
agents, such as, for example, cationic polypeptides and/or chelating agents
with quaternary ammonium
compounds or surfactants. The invention also teaches the use of synergistic
antimicrobial composition in
immersing or flushing or coating devices, such as catheters to inhibit biofilm
formation. The
compositions can also be incorporated into polymers which are used to form the
devices such as
catheters by impregnating or by drug-polymer conjugation.
[0035] The synergistic antimicrobial compositions require remarkably small
amounts of active
ingredients (compared to that which has been used in the fast) to be
effective. Because such small
amounts of active ingredients need to be used for these inventive synergistic
antimicrobial compositions,
the compositions are medically safe and environmentally friendly. These
compositions have properties
that include those of the separate compounds but go beyond them in efficacy
and scope of application.
9
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.._..~.~~w..~~.__ ._ _ _________._ _
CA 02452032 2003-12-04
The extremely low levels, and hence increased efficacy, of the active
compounds or ingredients make
this invention very desirable.
[0036] Novel compositions that combine iron-sequestering glycoprotein together
with cationic
polypeptides and/or chelating agents such that lesser quantities of iron-
sequestering glycoprotein,
cationic polypeptides and /or chelating agents than would normally be
necessary for an antimicrobial
composition are used to achieve significant biofilm inhibition. Higher
concentrations of these
compounds can be used if it is desired for certain applications.
(0037, The amount of iron-sequestering glycoprotein to be used in the
synergistic antimicrobial
composition of this invention can be between 125 to 2000 mg/L. The higher end
of this stated range
might be used to prepare a concentrated product that would be diluted prior to
use. For non-concentrated
products, the amount of iron-sequestering glycoprotein to be used in this
invention is preferably between
about 125 to 1000 mgJL.
[0038] The amount of cationic polypeptide to be used should be between about
12.5 to 200 mg/L.
The higher end of this range might apply if the compositions were formulated
as a concentrate. For non-
concentrated products, the amount of cationic polypeptide to be used in this
invention is preferably
between about 12.5-100 mg/L.
[0039] The amount of chelating agent to be used should be between about 12.5
to 200 mg/L. The
higher end of this range might apply if the compositions were formulated as a
concentrate. For non-
concentrated products, the amount of chelating agent to be used in this
invention is preferably between
about 12.5-100 mg/L.
(0040] By one method, if a three-component composition is to be formed
containing a chelating
agent, a cationic polypeptide and an iron-sequestering glycoprotein, these
compounds can be combined
in the following manner. With good stirring, a chelating agent can be
dissolved in water. A cationic
polypeptide can be added thereafter, followed by an iron-sequestering
glycoprotein. It should be noted,
however, that the addition order is not critical.
CA 02452032 2003-12-04
[0041] Since chelating agent and cationic polypeptide, such as, EDTA
(ethylenediaminetetraacetate)
and protamine sulfate, respectively are not readily soluble in water, they are
preferably added to the
composition in small amounts and stirred well. It may be necessary to adjust
the pH preferably to 7.4 in
order to make EDTA readily soluble.
[0042] Also, quaternary ammonium compounds and surfactants also may be
advantageously
combined with iron-sequestering glycoprotein in an antimicrobial composition.
A composition of the
invention comprises: (a) a small amount of at least one iron-sequestering
glycoprotein; and (b) a sparing
amount of at least one compound from the group consisting of a quaternary
ammonium compounds and
/or a surfactant, wherein, the amount of each of components (a) and (b) is
sufficient to form; in
combination, a synergistic antimicrobial composition.
[0043] The present invention includes adding an effective amount of
composition to the surface of an
object. This coating prevents the formation of biofilm on the surface, while
showing moderate effect on
the viability of microbes. The moderate effect on the viability of microbes
can be attributed to
biologically acceptable non-lethal compounds in the composition, which include
iron-sequestering
ovotransferrin and protamine sulfate. Lethal compounds such as silver or
antibiotics often create
selective pressure to increase the likelihood of amplifying silver-resistant
or antibiotic resistant strains,
thus rendering the antibiofilm agents useless. This is an important
consideration when the object to be
coated is a medical device that will be implanted in the body, where resident
bacteria exist. The
apparatus and method of the present invention uses composition to prevent
biofilm formation. The effect
of composition on biofilm formation by catheter-associated bacterial strains
on microtiter plates and on
vinyl urethral catheters was investigated.
[0044] Examples of bacteria that produce biofilms (biofilrn bacteria) which
can be inhibited by the
present invention include bacteria such as Staphylococcus epidermidis,
Enterococcus faecalis, E. eoli,
Proteus mirabilis, Pseudomonas aeruginosa, Klebsiella pneumaniae,
Staphylococcus aureus, and
Streptococcus viridans . These bacteria are commonly found associated with
medical devices including
catheters. Other bacteria producing biofilms which may be inhibited by the
compositions of the present
invention include Klebsiella oxytoca, Staphylococcus saprophyticus,
Providentia stuartii and Serratia
marcescens.
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CA 02452032 2004-08-04
[0045] Examples of devices that can be protected using the compositions of the
invention
include tubings and other medical devices, such as catheters, pacemakers,
prosthetic heart valves,
prosthetic joints, voice prostheses, contact lenses, intrauterine devices.
Medical devices include
disposable or permanent catheters, (e.g., central venous catheters, dialysis
catheters, long-term
tunneled central venous catheters, short-term central venous catheters,
peripherally inserted central
catheters, peripheral venous catheters, pulmonary artery Swan-Ganz catheters,
urinary catheters, and
peritoneal catheters), long-term urinary devices, tissue bonding urinary
devices, vascular grafts,
vascular catheter ports, wound drain tubes, ventricular catheters,
hydrocephalus shunts heart valves,
heart assist devices (e.g., left ventricular assist devices), pacemaker
capsules, incontinence devices,
penile implants, small or temporary joint replacements, urinary dilator,
cannulas, elastomers,
hydrogels, surgical instruments, dental instruments, tubings, such as
intravenous tubes, breathing
tubes, dental water lines, dental drain tubes, and feeding tubes, fabrics,
paper, indicator strips (e.g.,
paper indicator strips or plastic indicator strips), adhesives (e.g., hydrogel
adhesives, hot-melt
adhesives, or solvent-based adhesives), bandages, orthopedic implants, and any
other device used in
the medical field. Medical devices also include any device which may be
inserted or implanted into
a human being or other animal, or placed at the insertion or implantation site
such as the skin near
the insertion or implantation site, and which include at least one surface
which is susceptible to
colonization by biofilm embedded microorganisms. In one specific embodiment,
the composition of
the invention is integrated into an adhesive, such as tape, thereby providing
an adhesive which may
prevent growth or proliferation of biofilm embedded microorganisms on at least
one surface of the
adhesive. Medical devices for the present invention include surfaces of
equipment in operating
rooms, emergency rooms, hospital rooms, clinics, and bathrooms.
[0046] Implantable medical devices include orthopedic implants which may be
inspected for
contamination or infection by biofilm embedded microorganisms using endoscopy.
Insertable
medical devices include catheters and shunts which can be inspected without
invasive techniques
such as endoscopy. The medical devices may be formed of any suitable metallic
materials or non-
metallic materials known to persons skilled in the art. Examples of metallic
materials include, but
are not limited to, tivanium, titanium, and stainless steel, and derivatives
or combinations thereof.
Examples of non-metallic materials include, but are not limited to,
thermoplastic or polymeric
materials such as rubber, plastic, polyesters, polyethylene, polyurethane,
silicone, GortexTM
(polytetrafluoroethylene), DacronTM
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CA 02452032 2004-08-04
(polyethylene tetraphthalate), TeflonTM (polytetrafluoroethylene), latex,
elastomers and DacronTM
sealed with gelatin, collagen or albumin, and derivatives or combinations
thereof. The medical
devices include at least one surface for applying the composition of the
invention. Preferably, the
composition of the invention is applied to the entire medical device.
[0047] The composition of the invention may include any number of active
components and
base materials known to persons skilled in the art.
[0048) While the active components discussed herein may be 100% of the
composition of the
invention, preferably, the composition contains from at least about 0.01 % to
about 60% of the active
components by weight based upon the total weight of the composition of the
invention being
employed. In the preferred embodiment, the composition includes from at least
about 0.5% to about
30% (by weight) active components.
[0049] Other possible components of the composition include, but are not
limited to, buffer
solutions, phosphate buffered saline, saline, water, polyvinyl, polyethylene,
polyurethane,
polypropylene, silicone (e.g., silicone elastomers and silicone adhesives),
polycarboxylic acids, (e.g.,
polyacrylic acid, polymethacrylic acid, polymaleic acid, poly-(malefic acid
monoester), polyaspartic
acid, polyglutamic acid, aginic acid or pectimic acid), polycarboxylic acid
anhydrides (e.g.,
polymaleic anhydride, polymethacrylic anhydride or polyacrylic acid
anhydride), polyamines,
polyamine ions (e.g., polyethylene imine, polyvinylarnine, polylysine, poly-
(dialkylamineoethyl
methacrylate), poly-(dialkylaminomethyl styrene) or poly-(vinylpyridine)),
polyammonium ions
(e.g., poly-(2-methacryloxyethyl trialkyl ammonium ion), poly-(vinylbenzyl
trialkyl ammonium
ions), poly-(N.N.-alkylypyridinium ion) or poly-(dialkyloctamethylene ammonium
ion) and
polysulfonates (e.g. poly-(vinyl sulfonate) or poly-(styrene sulfonate)),
collodion, nylon, rubber,
plastic, polyesters, GortexTM (polytetrafluoroethylene), DacronTM
(polyethylene tetraphthalate),
TeflonTM (polytetrafluoroethylene), latex, and derivatives thereof, elastomers
and DacronTM (sealed
with gelatin, collagen or albumin, cyanoacrylates, methacrylates, papers with
porous barner films,
adhesives, e.g., hot melt adhesives, solvent based adhesives, and adhesive
hydrogels, fabrics, and
crosslinked and non-crosslinked hydrogels, and any other polymeric materials
which facilitate
dispersion of the active components and adhesion of the biofilm penetrating
coating to at least one
surface of the medical device. Linear
13
CA 02452032 2003-12-04
copolymers, cross-linked copolymers, graft polymers, and block polymers,
containing monomers as
constituents of the above exemplified polymers may also be used.
[0050] The term "effective" is herein defined as a sufficient amount of the
active components to
substantially prevent the growth or proliferation of biofilm embedded
microorganisms on the at least one
surface of the medical device in the case of the composition of the invention
being a coating; and as a
sufficient amount of the active components to substantially penetrate, or
break-up, the biofilm on the at
least one surface of the medical device, thereby facilitating access of the
active components,
antimicrobial agents, and/or antifungal agents to the microorganisms embedded
in the biofilrn, and thus,
removal of substantially all of the microorganisms from at least one surface
of the medical device in the
case of the composition of the invention being a solution. The amount will
vary for each of the active
components and upon known factors such as pharmaceutical characteristics; the
type of medical device;
the degree of biofilm embedded microorganism contamination; and the use and
length of use.
[0051 In another aspect, the invention is directed to a method for coating a
medical device. Broadly,
the method for coating a medical device includes the steps of providing a
medical device, providing, or
forming, a composition coating, and applying the composition coating to at
least one surface of the
medical device in an amount sufficient to substantially prevent the growth or
proliferation of biofilm
embedded microorganisms on at least one surface of the medical device.
[0052] In one specific embodiment, the method for coating a medical device
includes the steps of
forming a composition of the invention of an effective concentratian for
activating the active
components, and thus substantially preventing the growth or proliferation of
microorganisms on at least
one surface of the medical device, wherein the composition of the invention is
formed by combining a
active components and a base material. At least one surface of the medical
device is then contacted with
the composition of the invention under conditions wherein the composition of
the invention covers at
least one surface of the medical device. "Contacting" includes, but is not
limited to, impregnating,
compounding, mixing, integrating, coating, spraying and dipping.
[0053] In another aspect the invention relates to a method for inhibiting
biofilm embedded
microorganisms from at least one surface of the medical device. In one
specific embodiment, the method
14
CA 02452032 2003-12-04
of inhibiting biofilm from at least one surface of the medical device includes
the steps of providing a
medical device having at least one surface, the at least one surface having
biofilm attached thereto, and
contacting the medical device with a composition as described in greater
detail above. "Contacting"
further includes, but is not limited to, soaking, rinsing, flushing,
submerging, and washing. The medical
device should be contacted with the composition for a period of time
sufficient to remove substantially
all of the biofilm from the at least one surface of the medical device. In one
specific embodiment, the
medical device is submerged in the composition for at least 5 minutes.
Alternatively, the medical device
may be flushed with the composition. In the case of the medical device being a
tubing, such as dental
drain tubing, the composition may be poured into the dental drain tubing and
both ends of the tubing
clamped such that the composition is retained within the lumen of the tubing.
The tubing is then allowed
to remain filled with the composition for a period of time sufficient to
remove substantially all of the
microorganisms from at least one surface of the medical device, generally, for
at least about 1 minute to
about 48 hours. Alternatively, the tubing may be flushed by pouring the
composition into the lumen of
the tubing for an amount of time sufficient to prevent substantially all
biofilm growth.
[0054] The concentration of active components in the compositions may vary as
desired or necessary
to decrease the amount of time the composition of the invention is in contact
with the medical device.
These variations in active components concentration are easily determined by
persons skilled in the art.
[0055] In specific embodiments of the method for coating devices and the
methods for inhibiting
biofilm on at least one surface of the medical devices, the step of forming a
composition of the invention
may also include any one or all of the steps of adding an organic solvent, a
medical device material
penetrating agent, or adding an alkalinizing agent to the composition, to
enhance the reactivity of the
surface of the medical device with the composition. In the case of the method
for coating medical
devices, the organic solvent, medical device material penetrating agent,
and/or alkalinizing agent
preferably facilitate adhesion of the composition to at least one surface of
the medical device.
[0056] In another embodiment of the method for coating a medical device, the
composition coating is
preferably formed by combining a active components and a base material at room
temperature and
mixing the composition for a time sufficient to evenly disperse the active
agents in the composition prior
to applying the composition to a surface of the device. The medical device may
be. contacted with the
CA 02452032 2003-12-04
composition for a period of time sufficient for the composition to adhere to
at least one surface of the
device. After the composition is applied to a surface of the device, it is
allowed to dry.
[0057] The device is preferably placed in contact with the composition by
dipping the medical device
in the composition for a period of time ranging from about 5 seconds to about
120 minutes at a
temperature ranging from about 25°C to about 80°C. Preferably,
the device is placed in contact with the
composition by dipping the medical device in the composition for about 60
minutes at a temperature of
about 45°C. The device is then removed from the composition and the
composition is allowed to dry.
The medical device may be placed in an oven, or other heated environment for a
period of time
sufficient for the composition to dry.
[0058] Although one layer, or coating, of the composition is believed to
provide the desired
composition coating, multiple layers are preferred. The multiple layers of the
composition are preferably
applied to the at least one surface of the medical device by repeating the
steps discussed above.
Preferably, the medical device is contacted with the composition three times,
allowing the composition
to dry on at least one surface of the medical device prior to contacting the
medical device with the
composition for each subsequent layer. In other words, the medical device
preferably includes three
coats, or layers, of the composition on at least one surface of the medical
device.
[0059] In another embodiment, the method for coating medical devices with a
composition coating
includes the steps of forming a composition coating of an effective
concentration to substantially prevent
the growth or proliferation of biofilm on at least one surface of the medical
device by dissolving the
active components in an organic solvent, combining a medical device material
penetrating agent to the
active components and organic solvent, and combining an alkalinizing agent to
improve the reactivity of
the material of the medical device. The composition is then heated to a
temperature ranging from about
30°C to about 70°C to enhance the adherence of the composition
coating to at least one surface of the
device. The composition coating is applied to at least one surface of the
medical device, preferably by
contacting the composition coating to the at least one surface of the medical
device for a sufficient
period of time for the composition coating to adhere to at least one surface
of the medical device. The
medical device is removed from the composition coating and allowed to dry for
at least 8 hours, and
preferably, overnight, at room temperature. The medical device may then be
rinsed with a liquid, such as
16
CA 02452032 2004-08-04
water and allowed to dry for at least 2 hours, and preferably 4 hours, before
being sterilized. To
facilitate drying of the composition of the invention onto the surface of the
medical device, the
medical device may be placed into a heated environment such as an oven.
[0060] In another embodiment, the method for coating the medical devices with
a
composition includes the steps of forming the composition and incorporating
the composition into
the material forming the medical device during the formation of the medical
device. For example,
the composition may be combined with the material forming the medical device,
e.g., silicone,
polyurethane, polyethylene, GortexTM (polytetrafluoroethylene), DacronTM
(polyethylene
tetraphthalate), TeflonTM (polytetrafluoroethylene), and/or polypropylene, and
extruded with the
material forming the medical device, thereby incorporating the composition
into material forming the
medical device. In this embodiment, the composition may be incorporated in a
septum or adhesive
which is placed at the medical device insertion or implantation site. An
example of a coated medical
device having a composition incorporated into the material forming the medical
device in accordance
with this embodiment is the catheter insertion seal having an adhesive layer
described below in
greater detail.
[0061] In still another aspect, the invention is directed to coated medical
devices. Broadly,
the coated medical devices include a composition coating applied to at least
one surface of the
medical device. Suitable medical devices and compositions are described above
in greater detail.
The composition may be applied to at least one surface of the medical devices
in any suitable
manner. For example, the composition may be applied to the medical devices
following any of the
methods described above in greater detail.
EXAMPLE 1
Effects of ovotransferrin (OT), protamine sulfate (PS) and EDTA alone and in
combinations on
biofilm formation in catheter-associated bacteria
[0062] Catheter-associated bacterial strains used: E. coli P18, Proteus
mirabilis,
Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterococcus, f'aecalis and
Staphylococcus
epidermidis
17
CA 02452032 2003-12-04
[0063] Method: Base Formulas for Ovotranferrin (OT), Protamine Sulfate (PS)
and EDTA were
prepared as described in Table 1 below.
Table 1 Base Formulas for Screening (p,g/ml of sterile water)
Compound A B C D E
OT+PS 125+12.5 250+25 500+50 1000+100 2000+200
OT+EDTA 125+12.5 250+2S 500+50 1000+100 2000+200
PS+EDTA 12.5+12.5 25+25 50+SO 100+100 200+200
OT+PS+EDTA 125+12.5+I2.5 250+2S+25 500+50+50 1000+100+100 2000+200+200
j0064] Studies were done to test biofilm formation in microtiter plate wells.
Quantitative biofilm
assay for catheter-associated bacteria was standardized following the
procedure described by Jackson, et
al. (J. Bacteriol. 184: 290-301 ). Bacteria were routinely cultured at
37°C in Luria-Bertani (LB) or
Tryptic Soy Broth (TSB). Biofilm assays were generally carried out in colony-
forming antigen (CFA)
medium at 26°C. However, biofilm assays for Enterococcus faecalis and
Staphylococcus epidermidis
were carried out in TSB at 37°C.
[0065] Overnight cultures were inoculated 5:100 into fresh medium. In the
microtiter plate assay,
inoculated cultures were grown in a 96-well polystyrene microtiter plate.
Aqueous solutions of three
compounds ovotransferrin, protamine sulfate and EDTA were prepared separately
and appropriate
volume of each one was added to microtiter plate wells in replicates
individually and in combinations.
Concentrations of three compounds, ovotransferrin, protamine sulfate and EDTA
ranged from 0-2000
pg/ml and 0-200 pg/ml and 0-200 ug/ml, respectively. Growth of planktonic
cells was determined by
absorbance at 600 nm using Labsystems Multiskan Ascent microplate reader.
Biofilm was measured by
discarding the medium, rinsing the wells with water (three times) and staining
bound cells with crystal
violet. The dye was solubilized with 33% acetic acid, and absorbance at 630 nm
was determined using a
microtiter plate reader. For each experiment, background staining was
corrected by subtracting the
crystal violet bound to uninoculated control.
18
CA 02452032 2003-12-04
Results:
[0066] Figures 1-12 show biofilm formation by the above catheter-associated
bacterial strains in the
wells of microtiter plate in the presence and absence of ovotransferrin,
protamine sulfate and EDTA
alone and in combinations at different concentrations. Values represent the
Mean ~ Standard Deviation
of three experiments with four replicates for each concentration. The
composition consisting of all three
compounds showed synergistic inhibitory effects on biofilm formation in
Pseudomonas aeruginosa,
Klebsiella pneumoniae and Staphylococcus epidermidis (Figures 7-12).
Furthermore, ovotransfernn and
protamine sulfate together also showed synergistic inhibitory effects on
biofilm formation in Proteus
mirabilis, Klebsiella pneumoniae, Staphylococcus epidermidis and Pseudomonas
aeruginosa (Figures
1 _6).
EXAMPLE 2
Effects of synergistic composition on biofilm formation by catheter-associated
bacteria in urinary
catheter
[0067] Catheter-associated bacterial strains used: E. coli P18, Proteus
mirabilis, Pseudomonas
aeruginosa, Klebsiella pneumoniae, Enterococcus faecalis and Staphylococcus
epidermidis
Method:
[0068] To visualize biofilm formation in catheters, 35 ~,1 of an overnight
culture of each of the above
bacterial strain was inoculated into 700 p.l of medium and injected into clear
vinyl urethral catheters
overnight at 26 or 37°C with and without composition consisting of 1000
pg ovotransferrin/ml +100 p,g
protamine sulfate/ml+100 pg EDTA/ml. The catheters were capped at both ends.
The media and growth
conditions were as described above for microtiter plate assay. Cultures were
removed to determine the
growth at 600 nm and the catheters were rinsed with distilled water. After
drying at room temperature
for 15 min., 700 p,l of 1 % crystal violet was added to the catheters for 20
min. The stained biofilms were
rinsed several times with distilled water, and allowed to dry at room
temperature for 15 min before
examination. The dye was solubilized with 33% acetic acid, and absorbance at
630 nm was determined
using a spectrophotometer. The effect of composition on biofilm formation by
catheter-associated
19
CA 02452032 2003-12-04
bacterial strains such as Staphylococcus epidermidis, Proteus mirabilis and
Pseudomonas aeruginosa
was tested by growth of the organisms in urethral catheters as described
above:
Results:
[0069] Staphylococcus epidermidis and Proteus mirabilis formed biofilms mainly
at the air liquid
interface, while the biofilm formed by Pseudomonas aeruginosa was dispersed
all along the catheter.
The composition inhibited biofilm formation in all organisms (Fig. 13; Table
2).
Table 2: Biofilm formation by catheter-associated bacteria in urinary
catheters treated with synergistic
antimicrobial composition
I Or~anisrn Treated (OD) Untreated SOD) Inhibition (%)
Proteus mirabilis 0.0 0.10 100
Pseudomonas aeruginosa 0.20 0.96 79
Staphylococcus epidermidis O.OI 0.20 95
*Optical Density (OD) readings are based on crystal violet staining
EXAMPLE 3
[0070] Effect of synergistic composition on the viable cell counts of catheter-
associated bacteria
[0071] Catheter-associated bacterial strains used: E. coli P18, Proteus
mirabilis, Pseudomonas
aeruginosa, Klebsiella pneumoniae, Enterococcus faecalis and Staphylococcus
epideYmidis
Method:
]0072] Viable cell counts of catheter-associated bacterial strains were
determined following a
standard serial dilution plating method. The cultures of all six bacterial
strains were grown in the
presence and absence of a synergistic antimicrobial composition
(OT+pS+EDTA=1000+100+100
~,g/ml) using CFA medium (for gram-negative strains) and TSB (for gram-
positive strains) at 26°C and
37°C, respectively for 24 hours and serially diluted (10-fold dilution)
with sterile water. The number of
dilutions depended on the initial density of the cell suspension. Plated out
100 ~,1 of each dilution on LB
CA 02452032 2003-12-04
agar or Tryptic Soy agar plates in duplicates. The aliquot over the agar plate
surface was spread with a
sterile plastic spreader. The plates were incubated overnight at 37°C
and colony-forming units (CFU)
were counted and converted to original numbers/ml (CFU/ml) of suspension.
Further, viable cell counts
of treated cultures were compared with that of untreated ones (Table 3).
Results:
[0073] While the synergistic composition had dramatic effect on the viable
cell counts of E. coli and
Staphylococcus epideYmidis, it hardly showed any effect on that of other test
organisms. Interestingly,
when planktonic growth was measured in terms of turbidity on microtiter-plate
the synergistic
composition appeared to have minimal effect on the growth as compared to its
effect on biofilm
formation in all test organisms (see Example 1 ).
Table 3: Effect of synergistic composition on the viable cell counts of
catheter-associated bacteria
Organism Treated (CFU/ml) Untreated (CFU/ml) % Reduction
Escherichia coli P18 8.2x10' 4.6x1012 99.99
Proteus mirabilis 2.4x1014 8.4x1013 0.0
Klebsiella pneumoniae 7.1x1019 3.2x1018 0.0
Pseudomonas aeruginosa ND ND ND
Ente~ococcus mirabilis 1.6x1015 6.4x1014 0.0 ,
Staphylococcus epidermidis 1.Ox 10~ 1.6x 109 99.93
ND=Not Determined
EXAMPLE 4
Effect of coating urinary catheter with tridodecyl methyl ammonium chloride
(TDMAC)
plus synergistic antimicrobial composition on the growth of catheter-
associated bacteria
Catheter-associated bacterial strains used: Pseudomonas aeruginosa, Klebsiella
pneumoniae and Staphylococcus epidermidis
21
..,r._.x-.,-,. ,aa.._n .~.. , ~ x.~~.~4~,4fiXih%'s~v" ..~ 45&r~"~
~"..arro,~..c....~.,~"w,~~~._.._..~ v_.__..._.~~..~.....~.~.. ._ ~
.,~~.,a..~..._.P~.....,.,..a....._.__..._...-
CA 02452032 2003-12-04
Method:
Clear vinyl catheter segments (3 cm sections of tubing) that had been
preheated
(incubated in sterile water at 65°C overnight) were coated in 5% 'TDMAC
in ethanol for
an hour at room temperature. The catheter segments were vigorously washed with
sterile
water and air-dried. The segments were then immersed in ethanol and coating
solution
(1000 ~g OT+100 ~g PS +100 ~g EDTA/ml of water) for 2 hours at -20°C.
In addition, a
few catheter segments were immersed in coating solution alone for 2 hours at -
20°C. All
the segments were air dried and immersed in a tryptic soy broth culture of
Pseudomonas
aeruginosa, Klebsiella pneumoniae and Staphylococcus epidermidis for 3 hours
at 37°C.
The catheter segments were washed 3 times in 3 changes of sterile saline and
rolled on
tryptic soy agar plates. The plates were incubated overnight at 37°C
and the colonies
were counted. Proper controls were used as shown in the Table 4.
Results: See Table 4
Table 4: Effect of coating urinary catheter with TDMAC plus synergistic
antimicrobial
composition on the growth of catheter-associated bacteria
Bacterial Strain & Viable Counts (CFUlm I
Coatin h
ibition
%
n
Staph. epidermidis _
__
_
_
_
Control (uncoated) 16.8 ~ 3.3 0
Composition 13.2 ~ 0.6 21
TDMAC 20.4+7.7 0
TDMAC+Composition 11. Ot 1.3 3 5
ella pneumoniae
Control (uncoated) 3.80.9 0
Composition 2.3+1.8 40
TDMAC 4.3~ 1.3 0
TDMAC+Composition 1.60.6 59
monas aeruginosa
Control (uncoated) 21.211.5 0
Composition 15.31.3 28
TDMAC 18.84.6 12
TDMAC+Composition 9.80.5 54
*Colony Forming Units (CFU) per millimeter of catheter tubing
EXAMPLE 5
Effect of coating urinary catheter with polyvinylpyrrolidone (PVP) hydrogel
plus
synergistic antimicrobial composition on the growth of catheter-associated
bacteria
Catheter-associated bacterial strains used: Pseudomonas aeruginosa, Klebsiella
pneumoniae and Staphylococcus epidermidis
22
CA 02452032 2003-12-04
Method:
Clear vinyl catheter segments (3 cm sections of tubing) that had been
preheated
(incubated in sterile water at 65°C overnight) were immersed in PVP-
hydrogel (10%
PVP) and coating solution (1000 wg OT+100 ~g PS +100 ~g EDTA/ml of water) for
2
hours at 37°C. In addition, a few segments were immersed in coating
solution alone for 2
hours at 37°C. All the catheter segments were air dried and immersed in
a tryptic soy
broth culture of Pseudomonas aeruginosa, Klebsiella pneumoniae and
Staphylococcus
epidermidis for 3 hours at 37°C. The catheter segments were washed 3
times in 3 changes
of sterile saline and rolled on tryptic soy agar plates. The plates were
incubated overnight
at 37°C and the colonies were counted. Proper controls were used as
shown in the Table
S.
Results: See Table 5
Table 5: Effect of coating urinary catheter with PVP-Hydrogel plus synergistic
antimicrobial
composition on the growth of catheter-associated bacteria
Bacterial Strain & Viable Counts (CFUlmM) % Inhibition
Coating
Staph.epidermidis
Control (uncoated) 6.30.7 0
Composition 5.42.3 14
PVP-Hydrogel 5.8~ 1.1 8
PVP-Hydrogel+Composition 1.8~ 1.1 60
ella pneumonfae
Control (uncoated) 3.62.4 0
Composition 1.60.2 57
PVP-Hydrogel 1.50.6 59
PVP-Hydrogel+Composition 0.40.1 88
moms aeruginosa
Control (uncoated) 9.6+2.5 0
Composition 6.60.9 32
PVP-Hydrogel 8.2+2.3 15
PVP-Hydrogel+Composition 3.21.7 67
*Colony Forming Units (CFU) per millimeter of catheter tubing
[0074 Numerous modifications and variations of practicing the present
invention are possible in
light of the above teachings and therefore fall within the scope of the
following claims.
23
CA 02452032 2004-08-04
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