U.S. patent application number 13/609714 was filed with the patent office on 2014-06-19 for compositions and methods for the treatment and prevention of infections caused by staphylococcus aureus bacteria.
This patent application is currently assigned to University of Medicine and Dentistry of New Jersey. The applicant listed for this patent is Jeffrey B. Kaplan. Invention is credited to Jeffrey B. Kaplan.
Application Number | 20140170131 13/609714 |
Document ID | / |
Family ID | 40642197 |
Filed Date | 2014-06-19 |
United States Patent
Application |
20140170131 |
Kind Code |
A1 |
Kaplan; Jeffrey B. |
June 19, 2014 |
Compositions and Methods for the Treatment and Prevention of
Infections Caused by Staphylococcus aureus Bacteria
Abstract
The present invention relates to antimicrobial
deoxyribonuclease-based compositions that inhibit growth and
proliferation of Staphylococcus aureus bacteria. The present
invention also relates to methods of administering the compositions
in the treatment and prevention of S. aureus infections. The
present invention also relates to methods of administering the
compositions in the eradication of S. aureus nasal carriage, in
order to prevent the transmission of S. aureus bacteria
Inventors: |
Kaplan; Jeffrey B.; (Monsey,
NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kaplan; Jeffrey B. |
Monsey |
NY |
US |
|
|
Assignee: |
University of Medicine and
Dentistry of New Jersey
Somserset
NJ
|
Family ID: |
40642197 |
Appl. No.: |
13/609714 |
Filed: |
September 11, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12288198 |
Oct 17, 2008 |
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13609714 |
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Current U.S.
Class: |
424/94.61 |
Current CPC
Class: |
A01N 63/00 20130101;
A61K 38/465 20130101; A61P 31/04 20180101; C12Y 301/21001 20130101;
A01N 33/02 20130101; A61K 38/465 20130101; A61K 31/14 20130101;
A61K 2300/00 20130101; A61K 31/14 20130101; A61K 45/06 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
424/94.61 |
International
Class: |
A01N 63/00 20060101
A01N063/00; A01N 33/02 20060101 A01N033/02 |
Goverment Interests
[0002] This invention was made with government support under Grant
No. 5R01DE015124 awarded by National Institutes of Health. The
government has certain rights in the invention.
Claims
1-7. (canceled)
8. A method for increasing sensitivity of S. aureus
biofilm-embedded cells to killing, said method comprising treating
the S. aureus biofilm-embedded cells with a deoxyribonuclease
enzyme, or a deoxyribonuclease fragment or variant thereof followed
by treatment of the deoxyribonuclease enzyme treated cells with an
antimicrobial agent or mixture of antimicrobial agents.
9. The method of claim 8 wherein the antimicrobial agent or mixture
of antimicrobial agents is active against S. aureus cells.
10. The method of claim 8 wherein the deoxyribonuclease enzyme is
deoxyribonuclease I.
11. The method of claim 8 wherein the deoxyribonuclease enzyme is
bovine deoxyribonuclease I.
12. The method of claim 8 wherein the deoxyribonuclease enzyme is
human deoxyribonuclease I.
13. The method of claim 8 wherein the antimicrobial agent is a
quaternary ammonium compound.
14. The method of claim 13 wherein the quaternary ammonium compound
is cetylpyridinium chloride.
15. The method of claim 8 wherein the cells are treated with the
deoxyribonuclease enzyme for at least 10 minutes.
16. The method of claim 15 wherein the cells are treated with the
antimicrobial agent or mixture thereof for at least 3 minutes.
17. The method of claim 8 wherein the cells are treated with at
least 100 .mu.g/ml of the deoxyribonuclease enzyme.
Description
[0001] This patent application is a continuation of U.S.
application Ser. No. 12/288,198, filed Oct. 17, 2008, which claims
the benefit of priority from U.S. Provisional application No.
60/994,471, filed Oct. 18, 2007, teachings of each of which are
herein incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0003] The present invention relates to antimicrobial
deoxyribonuclease-based compositions that inhibit growth and
proliferation of Staphylococcus aureus bacteria. The present
invention also relates to methods of administering the compositions
in the treatment and prevention of S. aureus infections. The
present invention also relates to methods of administering the
compositions in the eradication of S. aureus nasal carriage, in
order to prevent the transmission of S. aureus bacteria.
BACKGROUND
[0004] The Gram-positive bacterium Staphylococcus aureus is a major
human pathogen (Lowy, 1998. New Engl. J. Med. 339:520-532). S.
aureus causes numerous infections including acute skin abscesses
(pimples, boils, styes, furunculosis) and invasive infections
(pneumonia, mastitis, phlebitis, meningitis, urinary tract
infections, osteomyelitis), as well as life-threatening bacteremias
and endocarditis. S. aureus is a major pathogen in nosocomial
infections, and in infections in patients with indwelling medical
devices. S. aureus is also a major pathogen in infections of
wounds, including infected diabetic foot ulcers, as well as in burn
wounds. S. aureus can also cause toxin-mediated infections
including food poisoning and toxic shock syndrome. Over the past 20
years, the frequencies of both nosocomial and community-acquired S.
aureus infections has been steadily increasing (Stevens, 2003.
Curr. Opin. Infect. Dis. 16:189-191). In addition, numerous
multidrug-resistant strains of S. aureus have emerged in recent
years (Bal & Gould, 2005. Expert Opin. Pharmacother.
6:2257-2269). These include methicillin-resistant S. aureus (MRSA),
which are resistant to all penicillinase-resistant penicillins and
cephalosporins (Lowy, 1998. New Engl. J. Med. 339:520-532).
Infections caused by MRSA are commonly treated with vancomycin
(Pope & Roecker, 2007. Expert Opin. Pharmacother. 8:1245-1261).
Recently, however, vancomycin-resistant S. aureus (VRSA) strains
have been isolated (Whitener et al., 2004. Clin. Infect. Dis.
38:1049-1055). In addition, S. aureus strains that exhibit
resistance to intermediate levels of vancomycin
(vancomycin-intermediate S. aureus or VISA) have been isolated
(Centers for Disease Control and Prevention, 1997. MMWR Morb.
Mortal. Wkly. Rep. 46:624-626). The percentage of S. aureus
infections caused by MRSA, VRSA and VISA strains has been
increasing (Lodise & McKinnon, 2007. Pharmacother.
27:2002-2012). Infections caused by MRSA, VRSA and VISA strains are
often more severe, more easily transmitted, and more difficult to
treat, than are infection caused by methicillin-sensitive S. aureus
(MSSA) strains (Tristan et al., 2007. J. Hosp. Infect. 65 Suppl
2:105-109). Also, multidrug-resistance may eventually lead to the
evolution of S. aureus strains that are resistant to all known
antibiotics. New methods for treating and preventing S. aureus
infections are urgently needed.
[0005] S. aureus is the leading cause of hospital-acquired
infections. The federal Centers for Disease Control and Prevention
estimates that in 2006 one in 22 hospitalized patients will
experience a hospital-acquired infection, resulting in a total of
1.7 million infections and 99,000 deaths (Sack, 2007. New York
Times July 27, p. 1). These nosocomial infections account for a
significant portion of healthcare expenditures in the United States
(Lodise & McKinnon, 2007. Pharmacother. 27:2002-2012). People
who are at a higher risk for S. aureus infections include
hospitalized patients, older patients, patients with type 1
diabetes, intravenous drug users, patients undergoing hemodialysis,
surgical patients, HIV patients, patients with intravascular
devices, patients with prosthetic heart valves, patients taking
immunosuppressive drugs, and patients with defective leukocyte
function. The large number of susceptible patients and the high
number of nosocomial infections and deaths underscores the need for
improved methods for treating and preventing S. aureus
infections.
[0006] S. aureus is a natural commensal bacterium that colonizes
the anterior nares of approximately 30 to 50 percent of healthy
adults. Infection results when a breach in the mucosal barrier or
skin allows bacterial cells access to the underlying tissues or to
the bloodstream (Lowy, 1998. New Engl. J. Med. 339:520-532). Sites
of infection are usually colonized by bacteria from the patient's
own nasal reservoir, from contact with an infected patient, or from
exposure to the transiently-colonized hands of healthcare workers.
Previous studies have shown that eradication of S. aureus nasal
carriage results in a decrease in the rate of S. aureus nosocomial
infections (Kallen et al., 2005. Infect. Control Hosp. Epidemiol.
26:916-922). Mupirocin cream, applied topically to the nares, has
been shown to effectively reduce S. aureus nasal carriage (Bertino,
1997. Amer. J. Health Systems Pharm. 54:2185-2191). However,
mupirocin cream needs to be administered 3 times per day for 5
days, and mupirocin-resistant MSSA and MRSA strains have been
identified (Kresken et al., 2004. Int. J. Antimicrob. Agents
23:577-581; Hurdle et al., 2005. J. Antimicrob. Chemother.
56:1166-1168). Therefore, there is a need for a method for
eradicating S. aureus nasal carriage that is more efficient and
less susceptible to the evolution of antimicrobial resistance.
[0007] S. aureus is known for its ability to form biofilms, which
are defined as communities of bacteria, encased in a
self-synthesized extracellular polymeric matrix, growing attached
to a biotic or abiotic surface (Gotz, 2002. Mol. Microbiol.
43:1367-1378). Evidence suggests that biofilm formation plays a
role in S. aureus wound infections (Akiyama et al., 1996. J.
Dermatol. Sci. 11:234-238) and osteomyelitis (Buxton et al., 1987.
J. Infect. Dis. 156:942-946). Biofilm formation may also play a
role in other localized S. aureus infections. Biofilms that form on
tissues or medical devices are extremely difficult to eradicate
because the biofilm mode of growth protects bacterial cells from
killing by antibiotics and host defenses (Fux et al., 2005. Trends
Microbiol. 13:34-40). Therefore, there is a need for anti-infective
therapies that can disperse S. aureus biofilms and kill
biofilm-embedded S. aureus bacteria.
SUMMARY OF THE INVENTION
[0008] In one embodiment, the present invention provides a
composition for preventing and/or inhibiting the growth of
biofilm-embedded S. aureus bacteria comprising: (a) a first
compound comprising a deoxyribonuclease, or an active fragment or
variant thereof, that disperses a biofilm; and (b) a second
compound comprising an antimicrobial agent that is active against
S. aureus cells.
[0009] In another embodiment, the deoxyribonuclease enzyme is
deoxyribonuclease I.
[0010] In another embodiment, the deoxyribonuclease enzyme is
bovine deoxyribonuclease I.
[0011] In another embodiment, the deoxyribonuclease enzyme is human
deoxyribonuclease I.
[0012] In yet another embodiment, the antimicrobial agent is the
quaternary ammonium compound cetylpyridinium chloride, also known
as hexadecylpyridinium chloride.
[0013] An embodiment of the invention includes a method for
treating a S. aureus infection by administering a composition
comprising (a) a deoxyribonuclease enzyme, or a deoxyribonuclease
fragment or variant thereof; and (b) an antimicrobial agent or
mixture of antimicrobial agents.
[0014] In yet another embodiment, the deoxyribonuclease-based
antimicrobial composition of the invention can be used to treat
various kinds of wounds, including, but not limited to, surgical
wounds, accidental wounds, burn wounds, leg ulcers, foot ulcers,
venous ulcers, diabetic ulcers, and pressure ulcers.
[0015] In yet another embodiment, the deoxyribonuclease-based
antimicrobial composition of the invention can be used to eradicate
S. aureus nasal carriage.
[0016] In yet another embodiment, the deoxyribonuclease-based
antimicrobial composition of the invention can be used to treat
ocular infections.
[0017] In yet another embodiment, the deoxyribonuclease-based
antimicrobial composition of the invention can be used as an
antiseptic rinse for use on skin, medical devices, surgical
instruments, and the like, before, during or after invasive
procedures such as catheter placement or surgery.
[0018] One aspect of the present invention includes providing
methods of using the deoxyribonuclease-based antimicrobial
composition of the invention in wound care devices, including, but
not limited to, a spray applicator.
[0019] An additional aspect of the present invention includes wound
care ointments, gels, and lotions comprising the
deoxyribonuclease-based antimicrobial compositions of the
invention, in addition to binders, wetting agents, adherents,
thickeners, stabilizers, fillers, and the like.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 shows that treatment of 24-h-old S. aureus biofilms
grown in microtiter plate wells with a solution of 100 .mu.g/ml of
deoxyribonuclease I (10 min treatment) causes significant
detachment of the biofilm, as judged by visual inspection of the
amount of crystal violet staining material remaining in the well
after treatment.
[0021] FIG. 2 shows that the detachment of 24-h-old S. aureus
biofilms grown in microtiter plate wells by deoxyribonuclease I (10
min treatment) is dependent on the deoxyribonuclease I
concentration, as judged by quantitation of the amount of crystal
violet stain remaining in the well after treatment (Absorbance at
595 nm).
[0022] FIG. 3 shows that a solution of 100 .mu.g/ml of
deoxyribonuclease I (10 min treatment) is capable of detaching
5-h-old, 8-h-old, 12-h-old and 24-h-old S. aureus biofilms grown in
microtiter plate wells.
[0023] FIG. 4 shows that S. aureus cells grown in medium
supplemented with 100 .mu.g/ml of deoxyribonuclease I exhibit much
less clumping (autoaggregation) than do cells grown in
unsupplemented medium.
[0024] FIG. 5 shows that S. aureus cells grown in tubes in medium
supplemented with 100 .mu.g/ml of deoxyribonuclease I exhibit much
less biofilm formation than do cells grown in unsupplemented
medium.
[0025] FIG. 6 shows that S. aureus cells grown in microplate wells
in medium supplemented with 100 .mu.g/ml of deoxyribonuclease I are
incapable of forming distinct biofilm colonies.
[0026] FIG. 7 shows that the inhibition of S. aureus biofilms grown
in microtiter plate wells by deoxyribonuclease I is dependent on
the deoxyribonuclease I concentration.
[0027] FIG. 8 shows that S. aureus biofilm cells grown in
microplate wells are resistant to killing by 100 .mu.g/ml of
deoxyribonuclease I for 10 min, and by 0.3% cetylpyridinium
chloride (CPC) for 5 min, but that treatment of the biofilms with
100 .mu.g/ml of deoxyribonuclease I for 10 min followed by
treatment with 0.3% CPC for 5 min results in significant killing of
the biofilm cells.
[0028] FIG. 9 shows that treatment of S. aureus biofilm cells grown
in microplate wells with 100 .mu.g/ml of deoxyribonuclease I for 10
min followed by treatment with 0.3% CPC for 3 min or 5 min results
in significant killing of the biofilm cells.
DETAILED DESCRIPTION OF THE INVENTION
[0029] This invention relates to a method and composition for
preventing and/or inhibiting the growth of biofilm-embedded S.
aureus bacteria. The basis of the invention is the discovery that
the bacteria is most susceptible when it is subject first to a
means of detaching S. aureus biofilm and then is exposed to an
agent which kills the bacteria.
[0030] It has been found that deoxyribonuclease enzyme or active
fragment or variant thereof is capable of inhibiting S. aureus
biofilm formation when added to a culture medium. Examples include
human deoxyribonuclease I and bovine deoxyribonuclease I.
[0031] Agents which are capable of killing S. aureus are known in
the art and include antimicrobial compounds such as quaternary
ammonium salts. Examples of quaternary ammonium salts include for
example, but not limited to, cetylpyridinium chloride,
methacryloyloxydodecyl pyridinimium bromide, like pyridinium halide
salts, benzalkoniumchloride, methacryloxylethylbenzyl
dimethylammonium chloride and methacryloxylethylcetyldimethyl
ammonium chloride.
[0032] The S. aureus can be treated by the administration of the
deoxyribonuclease enzyme and the antimicrobialanat agent at the
same time or serially with the deoxyribonuclease enzyme being
administered before the antimicrobial agent.
[0033] Any pharmaceutically acceptable vehicle or carrier, as well
as adjuvant, can be used in the manufacture, dissolution and
administration of pharmaceutical preparations of the invention
comprising deoxyribonuclease enzyme or active fragment or variant
thereof and/or the antimicrobial agent. Such vehicles, carriers and
adjuvants are well known to those of skill in the art and described
in text books such as Remington's Pharmaceutical Sciences, Mack
Publishing Co., Easton, Pa., 1985. Appropriate concentrations of
active composition to be incorporated into pharmaceutical
compositions can be routinely determined by those skilled in the
art and is dependent upon the form of administration as well as the
severity of the condition being treated.
[0034] Pharmaceutical formulations suitable for oral administration
may be provided in convenient unit forms including, but not limited
to, capsules or tablets, each containing a predetermined amount of
the deoxyribonuclease enzyme or active fragment or variant thereof
and/or the antimicrobial agent; as a powder or granules; as a
solution, a suspension or as an emulsion. The deoxyribonuclease
enzyme or active fragment or variant thereof and/or the
antimicrobial agent can also be presented as a bolus, electuary, or
paste. Tablets and capsules for oral administration may contain
conventional excipients such as binding agents, fillers,
lubricants, disintegrants, or wetting agents. The tablets may be
coated according to methods well known in the art. Timed release
formulations, which are known in the art, may also be suitable.
Oral liquid preparations may be in the form of, for example,
aqueous or oily suspensions, solutions, emulsions, syrups or
elixirs, or may be presented as a dry product for constitution with
water or other suitable vehicles before use. Such liquid
preparations may contain conventional additives such as suspending
agents, non-aqueous vehicles, including edible oils, or
preservatives.
[0035] Deoxyribonuclease enzyme or active fragment or variant
thereof and/or the antimicrobial agent of the present invention may
also be formulated for parenteral administration, such as by
injection, for example bolus injection or continuous infusion, and
may be provided in unit dose form in ampules, pre-filled syringes,
small volume infusion or in multi-dose containers with an added
preservative. Pharmaceutically acceptable compositions comprising a
deoxyribonuclease enzyme or active fragment or variant thereof
and/or the antimicrobial agent for parenteral administration may be
in the form of a suspension, solution or emulsion in oily or
aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing, and/or dispersing agents. Alternatively,
the active ingredient may be in powder form, obtained by asceptic
isolation of sterile solid or by lyophilization from solution, for
constitution with a suitable vehicle such as sterile, pyrogen free
water, before use.
[0036] For topical administration to the epidermis,
deoxyribonuclease enzyme or active fragment or variant thereof
and/or the antimicrobial agent of the present invention may be
formulated in an ointment, cream, or lotion, or as a transdermal
patch. Ointments and creams, may, for example, be formulated with
an aqueous or oily base with the addition of suitable thickening
and/or gelling agents. Lotions may be formulated with an aqueous or
oily base and will in general also contain one or more emulsifying
agents, stabilizing agents, suspending agents, thickening agents,
or coloring agents. Formulations suitable for topical
administration in the mouth include lozenges comprising
deoxyribonuclease enzyme or active fragment or variant thereof
and/or the antimicrobial agent in a flavored base, usually sucrose
and acacia or tragacanth; pastilles comprising the active
ingredient in an inert base such as gelatin and glycerin or sucrose
and acacia; and mouth washes comprising the active ingredient in a
suitable liquid carrier. For topical administration to the eye, the
deoxyribonuclease enzyme or active fragment or variant thereof
and/or the antimicrobial agent can be made up in solution or
suspension in a suitable sterile aqueous or non-aqueous vehicle.
Additives such as buffers (e.g. sodium metabisulphite or disodium
edeate) and thickening agents such as hypromellose can also be
included.
[0037] For intra-nasal administration, deoxyribonuclease enzyme or
active fragment or variant thereof and/or the antimicrobial agent
of the present invention can be provide in a liquid spray or
dispersible powder or in the form of drops. Drops may be formulated
with an aqueous or non-aqueous base also comprising one or more
dispersing agents, solubilizing agents, or suspending agents.
Liquid sprays are conveniently delivered from pressurized packs.
For administration by inhalation, deoxyribonuclease enzyme or
active fragment or variant thereof and/or the antimicrobial agent
of the present invention can be delivered by insufflator, nebulizer
or a pressurized pack or other convenient means of delivering the
aerosol spray. Pressurized packs may comprise a suitable propellant
such as dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount.
[0038] Alternatively, for administration by inhalation or
insufflation, the deoxyribonuclease enzyme or active fragment or
variant thereof and/or the antimicrobial agent of the present
invention can take the form of a dry powder composition, for
example a powder mix of the active component and a suitable powder
base such as lactose or starch. The powder composition may be
presented in unit dosage form in, for example, capsules, cartridges
or blister packs of gelatins, from which the powder can be
administered with the aid of an inhalator or insufflator.
[0039] When desired, any of the above-described formulations may be
adapted to provide sustained release of the deoxyribonuclease
enzyme or active fragment or variant thereof and/or the
antimicrobial agent.
[0040] The amount of deoxyribonuclease enzyme or active fragment or
variant thereof and/or the antimicrobial agent of the present
invention required for use in treatment will of course vary not
only with the particular protein or active fragment or variant
selected but also with the route of administration, the nature of
the condition being treated, and the age and condition of the
organism.
[0041] Increasing detachment of bacteria from a biofilm is also
expected to decrease resistance of the bacteria to antibiotic
therapy. Accordingly, the present invention also provide methods
for enhancing efficacy of antibiotic therapy against bacterial
infections by administration of a pharmaceutical composition of the
present invention in combination with or prior to administration of
an antibiotic.
[0042] In another embodiment of the present invention, wound
dressings including but not limited to sponges or gauzes can be
impregnated with the isolated deoxyribonuclease enzyme or active
fragment or variant thereof and/or the antimicrobial agent thereof
to prevent or inhibit bacterial or fungal attachment and reduce the
risk of wound infections. Similarly, catheter shields as well as
other materials used to cover a catheter insertion sites can be
coated or impregnated with a deoxyribonuclease enzyme or active
fragment or variant thereof and/or the antimicrobial agent to
inhibit bacterial or fungal biofilm attachment thereto. Adhesive
drapes used to prevent wound infection during high risk surgeries
can be impregnated with the isolated protein or active fragment or
variant thereof as well. Additional medical devices which can be
coated with a deoxyribonuclease enzyme or active fragment or
variant thereof and/or the antimicrobial agent thereof include, but
are not limited, central venous catheters, intravascular catheters,
urinary catheters, Hickman catheters, peritoneal dialysis
catheters, endotracheal catheters, mechanical heart valves, cardiac
pacemakers, arteriovenous shunts, schleral buckles, prosthetic
joints, tympanostomy tubes, tracheostomy tubes, voice prosthetics,
penile prosthetics, artificial urinary sphincters, synthetic
pubovaginal slings, surgical sutures, bone anchors, bone screws,
intraocular lenses, contact lenses, intrauterine devices,
aortofemoral grafts and vascular grafts. Exemplary solutions for
impregnating gauzes or sponges, catheter shields and adhesive
drapes or coating catheter shields and other medical devices
include, but are not limited to, phosphate buffered saline (pH
approximately 7.5) and bicarbonate buffer (pH approximately
9.0).
[0043] In yet another embodiment, an isolated deoxyribonuclease
enzyme or active fragment or variant thereof and/or the
anatimicrobial agent can be incorporated in a liquid disinfecting
solution. Such solutions may further comprise antimicrobials or
antifungals such as alcohol, providone-iodine solution and
antibiotics as well as preservatives. These solutions can be used,
for example, as disinfectants of the skin or surrounding area prior
to insertion or implantation of a device such as a catheter, as
catheter lock and/or flush solutions, and as antiseptic rinses for
any medical device including, but not limited to catheter
components such as needles, Leur-Lok connectors, needleless
connectors and hubs as well as other implantable devices. These
solutions can also be used to coat or disinfect surgical
instruments including, but not limited to, clamps, forceps,
scissors, skin hooks, tubing, needles, retractors, scalers, drills,
chisels, rasps and saws.
[0044] The compositions and method of the invention can be used for
the treatment and prevention of wound and burn infections caused by
S. aureus as well as other infections caused by S. aureus including
boils and sties and bovine mastitis. The compositions can be used
as a preprocedural rinse for surgery, as an antiseptic rinse, a
topical antiseptic and a catheter lock solution.
[0045] The composition and method of the instant invention can also
be used for the treatment and prevention of biofilm infections
caused by other bacteria including otitis media, sinusitis and
chronic obstructive pulmonary disease (Haemophilus influenzae),
dental caries (Streptococcus mutans), acne (Propionibacterium
acnes), and periodontitis (mixed-species biofilms).
EXAMPLES
Example 1
Deoxyribonuclease I Causes the Detachment and Dispersal of S.
aureus Biofilms
[0046] S. aureus Strain SH1000 (Horsburgh et al., 2002. J.
Bacteriol. 184:5457-5467) was used in all of the following
examples. The bacteria were passaged weekly on blood agar and
stored at 4.degree. C. Biofilms were cultured in Tryptic Soy broth
(Becton-Dickinson, Sparks, Md.) containing 6 g of yeast extract and
8 g of glucose per liter (TSB medium). All cultures were incubated
at 37.degree. C.
[0047] A biofilm formation assay was carried out as follows. A
loopful of cells from an agar plate was transferred to a
polypropylene microcentrifuge tube containing 200 .mu.l of TSB
medium. The cells were crushed with a disposable pellet pestle,
vortexed for 30 sec, diluted to 1 ml in fresh TSB medium, and then
passed through a 5-pm pore-size syringe filter to remove large
clumps of cells as previously described (Kaplan & Fine, 2002.
Appl. Environ. Microbiol. 68:4943-4950). Filtered cells were
diluted to 10.sup.3-10.sup.5 CFU/ml in TSB medium. Aliquots of
cells (200 .mu.l each) were transferred to the wells of a 96-well
tissue-culture-treated polystyrene microtiter plate (Falcon no.
324662, Becton-Dickinson) and the plate was incubated for 24 h. The
biofilms were rinsed once with water and then treated with 200
.mu.l of deoxyribonuclease I (bovine deoxyribonuclease I, purchased
from Sigma Chemical Company) at 100 .mu.g/ml in 150 mM NaCl, 1 mM
CaCl.sub.2. Control biofilms were treated with 200 .mu.l of 150 mM
NaCl, 1 mM CaCl.sub.2 alone. After 10 min at 37.degree. C.,
biofilms were rinsed with water and then dried. Biofilms were
stained for 1 min with 200 .mu.l of Gram's crystal violet stain
(catalog no. 23255960, Fisher Scientific, Fair Lawn, N.J.) and then
rinsed with water and dried. Previous studies showed that crystal
violet stains the bacterial biofilm biomass but not the polystyrene
microplate substrate (O'Toole & Kolter, 1998. Mol. Microbiol.
28:449-462).
[0048] FIG. 1 shows that the deoxyribonuclease I solution caused
the nearly complete detachment of the S. aureus biofilm from the
microplate well surface, as judged by the amount of crystal violet
staining material that remained in the well after treatment.
[0049] FIG. 2 shows the results of a similar experiment, except
that increasing amounts of deoxyribonuclease I were used, and the
amount of biofilm biomass remaining in the wells was quantitated by
destaining the biofilms for 10 min with 33% acetic acid (by vol)
and then measuring the absorbance of the crystal violet solution at
595 nm (A.sub.595). Concentrations of deoxyribonuclease I that were
less than 0.1 .mu.g/ml caused little detachment of the biofilm.
Concentrations of deoxyribonuclease I that were between 0.1 and 10
.mu.g/ml caused partial detachment of the biofilm. Concentrations
of deoxyribonuclease I that were greater than 10 .mu.g/ml caused
near complete detachment of the biofilm.
[0050] FIG. 3 shows the results of a similar experiment, except
that S. aureus biofilms that were grown for 5, 8, 12 or 24 h were
used. The concentration of deoxyribonuclease I was 100 .mu.g/ml and
the treatment time was 10 min. In this case, the amount of biofilm
biomass remaining in the well was quantitated by measuring the
A.sub.595 of the crystal violet stained biomass as described above,
and the percent of biofilm cell detachment was calculated using the
formula: 1-(A.sub.595[buffer+deoxyribonuclease I]/A.sub.595[buffer
alone]).times.100. As can be seen in FIG. 3, the deoxyribonuclease
I solution caused significant detachment of all of the S. aureus
biofilms, regardless of their age.
Example 2
Deoxyribonuclease I Inhibits S. aureus Autoaggregation and Biofilm
Formation
[0051] A series of experiments was performed in order to
demonstrate that deoxyribonuclease I inhibits S. aureus
autoaggregation and biofilm formation. These experiments were
carried out as described above, except that biofilms were grown in
16-mm.times.100-mm PET tubes (2 ml culture vol) in a rotary shaker
for 16 h. FIG. 4 shows S. aureus SH1000 cells cultured in this
manner in unsupplemented TSB medium formed large aggregates,
whereas cells cultured in this manner in TSB medium supplemented
with 100 .mu.g/ml of deoxyribonuclease I formed smaller aggregates.
Crystal violet staining of the culture tubes showed that
deoxyribonuclease I inhibited biofilm formation at the air-liquid
interface (FIG. 5).
[0052] FIG. 6 shows that SH1000 biofilms grown for 24 h in 96-well
microtiter plates in unsupplemented TSB medium formed distinct,
spherical colonies that were tightly attached to the microwell
surface, whereas biofilms grown in TSB medium supplemented with 100
.mu.g/ml of deoxyribonuclease I formed a dense film that uniformly
covered the microwell surface, but which readily detached after
gentle rinsing. FIG. 7 shows that deoxyribonuclease I inhibited
SH1000 biofilm formation in a dose-dependent manner, as determined
by measuring the A.sub.595 of the crystal violet stained biofilm
biomass as described above.
Example 3
Deoxyribonuclease I Increases the Sensitivity of S. aureus Biofilm
Cells to Killing by the Quaternary Ammonium Compound
Cetylpyridinium Chloride (CPC)
[0053] Biofilms were grown for 24 h in 96-well microtiter plates as
described above. Biofilms were rinsed once with water and then
treated with 200 .mu.l of TSB medium containing 100 .mu.g/ml of
deoxyribonuclease I. Control wells were treated with 200 .mu.l of
TSB medium alone. After 10 min at 37.degree. C., 20 .mu.l of 3% CPC
was added to each well and biofilms were incubated for 5 min at
room temperature. Control wells received 20 .mu.l of water. For
biofilms treated with TSB medium alone, biofilms were washed four
times with phosphate buffered saline to remove the CPC, and then
treated with 100 .mu.g/ml of deoxyribonuclease I to dissolve the
biofilm. This reaction was carried out in 100 .mu.g/ml in 150 mM
NaCl, 1 mM CaCl.sub.2 as described above. After 10 min, cells were
mixed and then serial dilutions were plated on agar. For S. aureus
biofilms treated with deoxyribonuclease I, cells were mixed and
then a 50-.mu.l aliquot of cells was diluted in 50 ml of phosphate
buffered saline. The cells were passed through an analytical test
filter funnel (no. 145-2020; Nalgene, Rochester, N.Y.), and the
filter was rinsed with 250 ml of sterile water, aseptically removed
from the filter unit, and placed on a blood agar plate. Colonies
were enumerated after 24 h.
[0054] As can be seen in FIG. 8, S. aureus biofilms treated with
either deoxyribonuclease I alone or CPC alone did not exhibit a
significant decrease in CFU/well values, whereas S. aureus biofilms
treated with deoxyribonuclease I followed by CPC exhibited an
approximately 4-log-unit decrease in CFU/well values. A significant
decrease in the CFU/well values was also observed after a 10 min
deoxyribonuclease I treatment followed by a 3 min CPC treatment
(FIG. 9).
* * * * *