U.S. patent application number 13/862154 was filed with the patent office on 2013-09-05 for methods and compositions for regulating biofilm development.
This patent application is currently assigned to NEWSOUTH INNOVATIONS PTY LIMITED. The applicant listed for this patent is NEWSOUTH INNOVATIONS PTY LIMITED. Invention is credited to Nicolas L. G. BARRAUD, Staffan KJELLEBERG, Scott A. RICE, Jeremy S. WEBB.
Application Number | 20130230606 13/862154 |
Document ID | / |
Family ID | 37451569 |
Filed Date | 2013-09-05 |
United States Patent
Application |
20130230606 |
Kind Code |
A1 |
BARRAUD; Nicolas L. G. ; et
al. |
September 5, 2013 |
METHODS AND COMPOSITIONS FOR REGULATING BIOFILM DEVELOPMENT
Abstract
The present disclosure relates to methods for promoting
dispersal of, or preventing formation of microbial biofilms,
including: exposing a biofilm to an effective amount of nitric
oxide or at least one nitric oxide generating or releasing agent;
treating a surface or medium susceptible to biofilm formation with
an effective amount of nitric oxide or at least one nitric oxide
generating or releasing agent; incorporating an effective amount of
nitric oxide or at least one nitric oxide generating or releasing
agent in a surface or medium susceptible to biofilm formation; or
inducing the accumulation of one or more reactive oxygen or
nitrogen species within microorganisms within said biofilm or
capable of forming a biofilm. The disclosure also relates to
methods for maintaining or enhancing or maintaining and enhancing
the functioning of a biofilm, comprising exposing a biofilm to at
least one nitric oxide scavenger, at least one antioxidant or at
least one nitric oxide scavenger and at least one antioxidant. The
disclosure also relates to compositions for promoting dispersal of,
or preventing formation of microbial biofilms, or for maintaining
or enhancing or maintaining and enhancing the functioning of
microbial biofilms.
Inventors: |
BARRAUD; Nicolas L. G.;
(Kingsford, AU) ; WEBB; Jeremy S.; (Coogee,
AU) ; RICE; Scott A.; (Paddington, AU) ;
KJELLEBERG; Staffan; (La Perouse, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NEWSOUTH INNOVATIONS PTY LIMITED |
Sydney |
|
AU |
|
|
Assignee: |
NEWSOUTH INNOVATIONS PTY
LIMITED
Sydney
AU
|
Family ID: |
37451569 |
Appl. No.: |
13/862154 |
Filed: |
April 12, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11915580 |
Sep 11, 2008 |
8425945 |
|
|
PCT/AU2006/000693 |
May 24, 2006 |
|
|
|
13862154 |
|
|
|
|
Current U.S.
Class: |
424/718 |
Current CPC
Class: |
Y02A 50/471 20180101;
A61K 33/40 20130101; A61K 33/26 20130101; A61K 38/063 20130101;
Y02A 50/473 20180101; A61P 31/10 20180101; A01N 41/12 20130101;
A01N 43/08 20130101; Y02A 50/475 20180101; Y02A 50/481 20180101;
A01N 37/44 20130101; A61K 33/00 20130101; A61P 43/00 20180101; C12N
11/16 20130101; A01N 43/50 20130101; A61K 31/197 20130101; Y02A
50/30 20180101; A01N 59/00 20130101; Y02A 50/478 20180101; A61P
31/04 20180101; A01N 59/16 20130101; A61K 45/06 20130101; A61K
31/375 20130101 |
Class at
Publication: |
424/718 |
International
Class: |
A01N 59/00 20060101
A01N059/00; A61K 45/06 20060101 A61K045/06; A61K 33/00 20060101
A61K033/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 24, 2005 |
AU |
2005902660 |
Claims
1. A method for promoting dispersal of, or preventing formation of
a microbial biofilm, the method comprising: exposing said biofilm
to an effective amount of nitric oxide or at least one nitric oxide
generating or releasing agent; treating a surface or medium
susceptible to biofilm formation with an effective amount of nitric
oxide or at least one nitric oxide generating or releasing agent;
incorporating an effective amount of nitric oxide or at least one
nitric oxide generating or releasing agent in a surface or medium
susceptible to biofilm formation; or inducing the accumulation of
an effective amount of one or more reactive oxygen or nitrogen
species within microorganisms within said biofilm or capable of
forming a biofilm.
2. The method of claim 1 wherein the at least one nitric oxide
generating or releasing agent comprises one or more nitric oxide
donors.
3. The method of claim 2 wherein the at least one nitric oxide
donor is sodium nitroprusside, S-nitroso-L-glutathione,
S-nitroso-N-acetylpenicillamine or a combination thereof.
4. The method of claim 1 wherein the microorganisms present in the
biofilm or capable of forming a biofilm are of a single
species.
5. The method of claim 1 wherein the microorganisms present in the
biofilm or capable of forming a biofilm are of multiple
species.
6. The method of claim 1, wherein the microorganisms within said
biofilm or capable of forming a biofilm comprise bacterial or
fungal species.
7. The method of claim 1, wherein the microorganisms within said
biofilm or capable of forming a biofilm comprise one or more
species selected from Candida spp., Hormoconis spp., Pseudomonas
spp., Pseudoalteromonas spp., Staphylococcus spp., Streptococcus
spp., Shigella spp., Mycobacterium spp., Enterococcus spp.,
Escherichia spp., Salmonella spp., Legionella spp., Haemophilus
spp., Bacillus spp., Desulfovibrio spp., Shewanella spp., Geobacter
spp., Klebsiella spp., Proteus spp., Aeromonas spp., Arthrobacter
spp., Micrococcus spp., Serratia spp., Porphyromonas spp.,
Fusobacterium spp. and Vibrio spp..
8. The method of claim 1, wherein the microorganisms within said
biofilm or capable of forming a biofilm comprise one or species
selected from P. aeruginosa, Staphylococcus epidprmidis,
Escherichia coli, Bacillus licheniformis, Serratia marcescens,
Fusobacterium nucleatum, Vibrio Cholerae, and Candida albicans.
9. The method of claim 1, further comprising treating said surface
or medium with, incorporating in said surface or medium, or
exposing the microorganisms within said biofilm or capable of
forming a biofilm to, at least one antimicrobial agent.
10. The method of claim 9 wherein the antimicrobial agent is
selected from an antibiotic, a surfactant, an oxidative
stress-inducing agent, or a combination thereof.
11. The method of claim 1 wherein the one or more reactive oxygen
or nitrogen species are selected from peroxynitrite, nitric oxide,
hydrogen peroxide and superoxide radicals, or a combination
thereof.
12. The method of claim 1 wherein said method comprises inducing
differentiation events in microorganisms within said biofilm which
lead to dispersal or wherein said method comprises preventing
induction of differentiation events in microorganisms which lead to
biofilm formation.
13. The method of claim 1 which comprises increasing the
sensitivity of a microorganism to one or more antimicrobial
agents.
14. The method of claim 1, wherein the effective amount comprises a
concentration of at least one nitric oxide generating or releasing
agent of from about 1 nM to about 10 mM.
15. The method of claim 1, wherein the effective amount comprises a
concentration of at least one nitric oxide generating or releasing
agent of from about 10 nM to about 5 .mu.M.
16. The method of claim 1, wherein the effective amount comprises a
concentration of at least one nitric oxide generating or releasing
agent of from about 10 nM to about 1 .mu.M.
17. The method of claim 1 comprising administering to a subject an
effective amount of nitric oxide or at least one nitric oxide
generating or releasing agent for the treatment or prevention of a
biofilm-associated condition in said subject.
18. The method of claim 17 further comprising administering to said
subject at least one antimicrobial agent.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/915,580, filed Nov. 26, 2007, which is the
U.S. National Stage Entry of PCT/AU2006/000693, filed May 24, 2006,
which claims priority to Australian Pat. App. No. 2005902660 filed
May 24, 2005, each of which is hereby incorporated by reference in
the present disclosure in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to methods and compositions
for regulating programmed cell death in microorganisms and for
promoting or inhibiting dispersal of microorganisms from
biofilms.
BACKGROUND OF THE INVENTION
[0003] Biofilms are three dimensional microbial growth forms
comprising bacterial communities and the extracellular matrix they
produce. Biofilms are ubiquitous in the environment and may form on
solid surfaces where water is available or in suspension, for
example as floes or granules. Biofilms cause significant industrial
damage, causing, for example, fouling and corrosion in fluid
processes such as water distribution and treatment systems, pulp
and paper manufacturing systems, heat exchange systems and cooling
towers, and contributing to the souring of oil in pipelines and
reservoirs. From a public health perspective, biofilms are also
important reservoirs of pathogens in water systems such as drinking
water, reservoirs and pipes. Biofilms are also associated with a 1
s number of chronic infections in humans, for example otitis media
(biofilms on surfaces of the ear), bacterial endocarditis (biofilms
on surfaces of the heart and heart valves), cystic fibrosis
(biofilms on surfaces of the lungs) and kidney stones, and readily
form on medical equipment such as implantable medical devices.
[0004] However notwithstanding the significant detrimental effects
of biofilms in many environments, biofilms may also be of benefit.
For example, in waste water treatment systems suspended floc
biofilms or surface-associated biofilms on membranes are said to
facilitate nutrient removal, such as in denitrification.
[0005] Accordingly, there is a clear need both for effective
strategies to eliminate deleterious biofilms and to enhance the
activity of beneficial biofilms.
[0006] Biofilms are essentially multicellular microbial
communities, the formation and development of which is dependent on
various multicellular traits of the member organisms, such as
cell-cell signalling. Extracellular signalling systems such as
quorum sensing are used by bacteria to assess cell density and
initiate changes in gene expression and phenotypes when sufficient
concentrations of signalling molecules are reached. This is
associated with differential gene expression, leading to the
induction of, for example, virulence factors and/or defence
mechanisms, and with cell differentiation such that
biofilm-associated cells become highly differentiated from
free-living (planktonic) cells.
[0007] As the cells within biofilms differentiate and biofilms
mature, reduced metabolic rates, the cellular expression of defence
mechanisms and the reduced ability of antimicrobial agents to
penetrate the biofilm results in increased antimicrobial resistance
and make biofilms particularly difficult to eradicate. Present
biofilm control strategies typically target the early stages of
biofilm development and involve the use of toxic antimicrobial
agents. However such toxic agents present their own downstream
problems due to their release into the environment. Improved
strategies for biofilm control are clearly required.
[0008] It has recently been discovered that Pseudomonas aeruginosa
cells within biofilms undergo programmed cell death and lysis in
the normal course of the biofilm lifecycle (Webb of al, 2003, Cell
death in Pseudomonas aeruginosa biofilm development, J. Bact., 185:
4585-4592). It is believed that programmed cell death in biofilms
of P. aeruginosa is prophage-mediated and plays a role in
facilitating differentiation and dispersal of a subpopulation of
surviving cells from the biofilm.
[0009] The present invention is based on the inventors' finding
that this phenomenon of programmed cell death in biofilms is linked
to the accumulation of reactive oxygen and nitrogen species (RONS)
within organisms of the biofilm and that the process of programmed
cell death, and dispersal of cells from a biofilm into planktonic
cells, can be induced using nitric oxide generators. The ability to
increase nitric oxide concentrations in vivo enables the regulation
and manipulation of biofilm developmental processes, by promoting
programmed cell death, and increases the sensitivity of the cells
to antimicrobial agents, thereby providing avenues for inhibiting
and/or reversing biofilm development.
SUMMARY OF THE INVENTION
[0010] The present invention relates to a method for promoting
dispersal of microorganisms from a biofilm, the method comprising
exposing the biofilm to an effective amount of nitric oxide or at
least one nitric oxide generating or releasing agent.
[0011] The present invention also relates to a method for promoting
dispersal of microorganisms from a biofilm, the method comprising
inducing the accumulation of one or more reactive oxygen and
nitrogen species within the microorganisms.
[0012] The present invention also relates to a method for
inhibiting biofilm formation and/or development, the method
comprising treating a surface or other medium susceptible to
biofilm formation an effective amount of nitric oxide or at least
one nitric oxide generating or releasing agent.
[0013] Thus, according to a first aspect of the present invention,
there is provided a method for promoting dispersal of, or
preventing formation of a microbial biofilm, the method comprising:
[0014] exposing said biofilm to an effective amount of nitric oxide
or at least one nitric oxide generating or releasing agent; [0015]
treating a surface or medium susceptible to biofilm formation with
an effective amount of nitric oxide or at least one nitric oxide
generating or releasing agent; [0016] incorporating an effective
amount of nitric oxide or at least one nitric oxide generating or
releasing agent in a surface or medium susceptible to biofilm
formation; or [0017] inducing the accumulation of one or more
reactive oxygen or nitrogen species within microorganisms within
said biofilm or capable of forming a biofilm.
[0018] The at least one nitric oxide generating or releasing agent
may comprise one or more nitric oxide donors. Said one or more
nitric oxide donors may be selected from sodium nitroprusside,
S-nitroso-L-glutathione, S-nitroso-N-acetylpenicillamine or a
combination thereof.
[0019] Typically the nitric oxide donor is provided in a non-toxic
concentration. For example, the concentration may be in the
nanomolar, micromolar or millimolar range, such as from about 1 nM
to about 10 mM or from about 10 nM to about 5 .mu.M.
[0020] The biofilm may be surface-associated or suspended. The
suspended biofilm may be in the form of flocs or granules.
[0021] The microorganisms present in the biofilms or capable of
forming biofilms may be of a single species or of multiple
species.
[0022] The microorganisms within said biofilm or capable of forming
a biofilm may comprise bacterial or fungal species or both, and may
comprise one or more species selected from, for example, Candida
spp., Hormoconis spp., Pseudomonas spp., Pseudoalteromonas spp.,
Staphylococcus spp., Streptococcus spp., Shigella spp.,
Mycobacterium spp., Enterococcus spp., Escherichia spp., Salmonella
spp., Legionella spp., Haemophilus spp., Bacillus spp.,
Desulfovibrio spp., Shewanella spp., Geobacter spp., Klebsiella
spp., Proteus spp., Aeromonas spp., Arthrobacter spp., Micrococcus
spp., Serratia spp., Porphyromonas spp., Fusobacterium spp. and
Vibrio spp., representative examples of such species being Candida
albicans, P. aeruginosa, Staphylococcus epidermidis, Escherichia
coil, Bacillus licheniformis, Serratia marcescens, Fusobacterium
nucleatum, and Vibrio Cholerae.
[0023] The method may further comprise treating the surface or
medium with, incorporating in said surface or medium, or exposing
the microorganisms within said biofilm or capable of forming a
biofilm to, at least one antimicrobial agent. By way of example,
the antibiotic may be an aminoglycoside such as tobramycin, the
surfactant may be sodium dodecyl sulfate and the oxidative
stress-inducing agent may be hydrogen peroxide, hypochlorous acid,
chlorine or chloramine.
[0024] Reactive oxygen and nitrogen species which may accumulate in
microorganisms of a biofilm or capable of forming biofilms treated
by a method of the invention may include peroxynitrite, nitric
oxide, hydrogen peroxide and superoxide radicals.
[0025] In one embodiment the reactive oxygen and nitrogen species
is peroxynitrite. Accumulation of reactive oxygen and nitrogen
species may be achieved by exposing the biofilm to an effective
amount of nitric oxide or at least one nitric oxide generating or
releasing agent.
[0026] Methods of the invention for promoting dispersal of or
preventing formation of biofilms may comprise inducing
differentiation events in microorganisms within said biofilm which
lead to dispersal or may comprise preventing induction of
differentiation events in microorganisms which lead to biofilm
formation. Alternatively, or as well, methods of the invention may
comprise increasing the sensitivity of a microorganism to
antimicrobial agents.
[0027] The present invention also relates to a method for the
treatment and/or prevention of a condition associated with biofilm
development, comprising administering to a subject an effective
amount of nitric oxide or at least one nitric oxide generating or
releasing agent.
[0028] Thus, a method of the invention for promoting dispersal of
or preventing formation of biofilms may comprise administering to a
subject an effective amount of nitric oxide or at least one nitric
oxide generating or releasing agent for the treatment or prevention
of a biofilm-associated condition in said subject, optionally
together with at least one antimicrobial agent.
[0029] The agent and/or the antimicrobial agent may be coated onto
or be impregnated in or incorporated in the surface of a suitable
medical device such as a catheter, stent, prosthesis or other
surgical or implantable device.
[0030] The present invention also relates to compositions for
promoting dispersal of microorganisms from a biofilm, or for
inhibiting biofilm formation and/or development.
[0031] Thus, according to another aspect of the invention, there is
provided a composition for promoting dispersal of, or preventing
formation of a microbial biofilm, the composition comprising nitric
oxide, at least one nitric oxide generating or releasing agent or
nitric oxide and a nitric oxide generating or releasing agent,
together with a suitable carrier.
[0032] The at least one nitric oxide generating or releasing agent
may comprise one or more nitric oxide donors. Said one or more
nitric oxide donors may be selected from sodium nitroprusside,
S-nitroso-L-glutathione, S-nitroso-N-acetylpenicillamine or a
combination thereof.
[0033] In one embodiment the nitric oxide donor is sodium
nitroprusside.
[0034] In particular embodiments the composition may be an
anti-fouling composition, a medical device or component thereof, a
coating for a medical device or a pharmaceutical composition.
[0035] The composition may further comprise at least one
antimicrobial agent. The antimicrobial agent may be any
antimicrobial agent such as an antibiotic, a surfactant or
oxidative stress-inducing agent. By way of example, the antibiotic
may be an aminoglycoside such as tobramycin, the surfactant may be
sodium dodecyl sulfate and the oxidative stress-inducing agent may
be hydrogen peroxide, hypochlorous acid, chlorine or
chloramine.
[0036] A composition of the invention for promoting dispersal of,
or preventing formation of a microbial biofilm, may: induce
differentiation events in microorganisms within said biofilm which
lead to dispersal or prevent induction of differentiation events in
microorganisms which lead to biofilm formation; increase the
sensitivity of said microorganisms to antimicrobial agents; or may
provide a combination of these effects.
[0037] The present invention also relates to compositions for
treating and/or preventing a condition associated with biofilm
development.
[0038] Thus, a composition of the invention for promoting dispersal
of, or preventing formation of a microbial biofilm may be suitable
for treating or preventing a biofilm-associated condition of a
subject, and may optionally comprise at least one antimicrobial
agent.
[0039] The present invention also relates to methods for
maintaining and/or enhancing the functioning of a biofilm,
comprising exposing the biofilm to at least one nitric oxide
scavenger and/or at least one antioxidant.
[0040] Thus, according to another aspect of the invention, there is
provided a method for maintaining or enhancing the functioning of a
biofilm, the method comprising exposing the biofilm to at least one
nitric oxide scavenger, at least one antioxidant or at least one
nitric oxide scavenger and at least one antioxidant.
[0041] In one embodiment, the nitric oxide scavenger is
2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide.
[0042] The antioxidant may be selected from the group consisting
of: thioredoxin, superoxide dismutase, glutathione and ascorbic
acid.
[0043] The method may comprise inhibiting differentiation events in
microorganisms within said biofilm which lead to dispersal.
[0044] According to another aspect of the present invention there
is provided a composition for maintaining or enhancing, or
maintaining and enhancing the functioning of a biofilm, the
composition comprising at least one nitric oxide scavenger and/or
at least one antioxidant together with a suitable carrier.
[0045] The composition may inhibit differentiation events in
microorganisms within said biofilm which lead to dispersal.
Definitions
[0046] As used herein the term "biofilm" refers to any
three-dimensional, matrix-encased microbial community displaying
multicellular characteristics. Accordingly, as used herein, the
term biofilm includes surface-associated biofilms as well as
biofilms in suspension, such as flocs and granules. Biofilms may
comprise a single microbial species or may be mixed species
complexes, and may include bacteria as well as fungi, algae,
protozoa, or other microorganisms.
[0047] As used herein the term "surface" includes both biological
surfaces and non-biological surfaces. Biological surfaces typically
include surfaces both internal (such as tissues and membranes) and
external (such as skin, seeds, plant foliage) to an organism,
including bacterial membranes and cell walls. Biological surfaces
also include other natural surfaces such as wood or fibre. A
non-biological surface may be any man made surface of any
composition that supports the establishment and development of a
biofilm. Such surfaces may be present in industrial plants and
equipment. Further, for the purposes of the present invention, a
surface may be porous (such as a membrane) or non-porous, rigid or
flexible.
[0048] As used herein the term "dispersal" as it relates to a
biofilm means the process of detachment of cells from surfaces,
including other cells (such as, one another, biofilm(s)), and a
return to a planktonic phenotype or behaviour of those cells.
[0049] As used herein the term "programmed cell death" means a
developmental event within a biofilm that occurs at defined stages
and causes autolysis, cellular differentiation and the development
of subpopulations of cells with specific phenotypes.
[0050] As used herein the term "exposing" means administering to,
or otherwise bringing into contact with. A microorganism or biofilm
may be exposed to an active agent directly or indirectly. Typically
direct exposure refers to administration of the agent to the
microorganism or biofilm to be treated or otherwise bringing the
microorganism or biofilm into contact with the agent itself.
Typically indirect exposure refers to the administration of a
precursor of the active agent or a compound or molecule capable of
generating, either solely or in reaction with other compounds or
molecules, the active agent to the microorganism or biofilm or
otherwise bringing the microorganism or biofilm into contact
therewith. Similarly, the terms "treat" and "treating" and
variations thereof as used herein mean administering to, or
otherwise bringing into contact with.
[0051] As used herein the term "effective amount" includes within
its meaning a non-toxic but sufficient amount or concentration of
an agent to provide the desired effect. The exact
amount/concentration required will vary depending on factors such
as the species of microorganism(s) being treated, the extent,
severity and/or age of a biofilm being treated, whether the biofilm
is surface-associated or suspended, the particular agent(s) being
administered and the mode of administration and so forth. Thus, it
is not possible to specify an exact "effective amount". However,
for any given case, an appropriate "effective amount" may be
determined by one of ordinary skill in the art using only routine
experimentation.
[0052] As used herein the term "non-toxic" as it pertains to
concentrations or amounts of a substance means concentrations or
amounts of a substance which do not have a direct toxic effect on
cells, do not kill individually free-living cells, but which may
operate as a signal that triggers induction of differentiation
processes in biofilms, which involve a programmed cell death
response and which therefore may result in death of a subpopulation
of cells, generation of dispersal cells and the dispersal of
biofilms. For example, with reference to nitric oxide donors, a
non-toxic concentration or amount may comprise 100 mM nitric oxide
donor, or less.
[0053] As used herein the term "functioning" as it pertains to a
biofilm may be measured with reference to any one or more of the
following parameters: the viability of the microorganisms in the
biofilm, the activity(ies) of the microorganisms in the biofilm,
the density of the microorganisms in the biofilm, the lifespan of
the biofilm and the efficacy of the biofilm in performing a
specific function, such as in the case of biofilms in wastewater
systems, the removal of nutrients. Accordingly, in the context of
the present invention "maintaining" biofilm functioning means
preventing or at least substantially reducing the developmental
processes of programmed cell death and dispersal such that
microorganism viability, microorganism activity, biofilm lifespan
and or biofilm function is not significantly reduced. "Enhancing"
biofilm functioning means increasing or improving any one or more
of the above parameters in comparison to a biofilm not treated in
accordance with the present invention.
[0054] As used herein the term "inhibiting" as it pertains to
biofilms means complete or partial inhibition of biofilm formation
and/or development and also includes within its scope the reversal
of biofilm development or processes associated with biofilm
formation and/or development. Further, inhibition may be permanent
or temporary. In terms of temporary inhibition, biofilm formation
and/or development may be inhibited for a time sufficient to
produce the desired effect.
[0055] In the context of this specification, the term "comprising"
means "including principally, but not necessarily solely".
Furthermore, variations of the word "comprising", such as
"comprise" and "comprises", have correspondingly varied
meanings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0056] The present invention will now be described, by way of
example only, with reference to the following drawings.
[0057] FIG. 1. Cell death and dispersal events in P. aeruginosa
biofilms correlate with the accumulation of reactive oxygen and
nitrogen species within microcolony structures. (A) Confocal
micrograph showing a 7 day biofilm stained with the BacLight
LIVE/DEAD stain (micrographs for live and dead cells are also shown
independently in separate panels). White arrow indicates a hollow
structure within a biofilm. Black arrow indicates dead cells within
a biofilm. Bar, 50 .mu.m. (B) Confocal micrographs of microcolonies
in a 7 day biofilm. Biofilms were stained with fluorescent dyes
detecting specific RONS. Left panel are phase contrast images,
right panel are fluorescence images showing RONS accumulation in XY
(top down) and XZ (side-on) views, Bars, 50 .mu.m. Images are
representative of at least 3 independent experiments.
[0058] FIG. 2. The effect of administration of SNP on biofilm and
planktonic growth of P. Aeruginosa. PAO1 cells were grown for 24
hours in 96-well plates in the presence of sodium nitroprusside
(SNP). Planktonic growth was quantified by fluorescence measurement
and biofilm formation by crystal violet staining. Control, no SNP
added.
[0059] FIG. 3. The effect of nitric oxide generating or releasing
agents on the transition from biofilm to planktonic growth in P.
aeruginosa. PAO1 cells were grown for 24 hours in petri dishes
containing microscope slides in the presence of a nitric oxide
donor, sodium nitroprusside (SNP), S-nitroso-L-glutathione (GSNO)
or S-nitroso-N-acetylpenicillamine (SNAP) or SNP plus the nitric
oxide scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl
3-oxide (PTIO). Planktonic growth was quantified by measuring the
optical density (OD.sub.600nm) of the supernatant (light bars) and
biofilm growth by measuring the percentage of surface coverage
(dark bars) using image analysis of digital photomicrographs of
microscope slides after staining.
[0060] FIG. 4. The effect of SNP treatment on the sensitivity of P.
aeruginosa to anti-microbial agents. P. aeruginosa PAO1 was grown
for 24 hours in petri dishes containing microscope slides in the
presence or absence of 500 nM SNP. Biofilms on microscope slides
were treated for 30 minutes with the antimicrobial solutions,
stained with LIVE/DEAD staining to allow analysis with light
microscopy and quantified (percentage surface coverage) using
digital image analysis. (A) Sensitivity of biofilms (% surface
coverage) on microscope slides to antimicrobials tobramycin (Tb),
hydrogen peroxide (H.sub.2O.sub.2), sodium dodecyl sulphate (SDS)
and hypochlorous acid (HOCl) in the presence (light grey bars) or
absence (dark grey bars) of SNP. (B) Confocal fluorescence
micrographs of the same slides after treatment.
[0061] FIG. 5. The effect of SNP treatment on the sensitivity of
pre-established P. aeruginosa biofilms to anti-microbial agents. P.
aeruginosa PAO1 was grown for 24 hours in petri dishes containing
microscope slides for 24 hours prior to the addition of 500 nM SNP.
Biofilms were grown for a further 24 hours and then treated for 30
minutes with the antimicrobial solutions, stained with LIVE/DEAD
staining to allow analysis with light microscopy and quantified
(percentage surface coverage) using digital image analysis. (A)
Sensitivity of biofilms (% surface coverage) on microscope slides
to tobramycin (Tb), hydrogen peroxide (H.sub.2O.sub.2) and
ultraviolet light (UV) in the presence (light grey bars) or absence
(dark grey bars) of SNP. (B) Confocal fluorescence micrographs of
the same slides after treatment.
[0062] FIG. 6. Combination treatment of planktonic P. aeruginosa
cells using SNP and anti-microbial agents. Planktonic cells were
treated for 2 hours with the antimicrobial agent tobramycin (Tb) or
hydrogen peroxide (H.sub.2O.sub.2). Plate counts of colony forming
units (logCFU/mL) were performed to assess the viability of the
bacteria.
[0063] FIG. 7. Effect of SNP exposure on dispersal in floc biofilms
from a wastewater treatment sludge reactor. Confocal fluorescence
micrographs of 13 day mixed species floc biofilms either untreated
(A) or treated with 10 mM SNP (B).
[0064] FIG. 8. Drinking water biofilms spiked with S.
marcescens--effect of SNP on the biofilms after 18 hours exposure
to 0, 100 nM or 500 nM SNP: (A) viability counts on oligotrophic
agar (R2A, triplicates); (B) viability counts on eutrophic agar
(LB.sub.10, triplicates); (C) percentage of surface coverage using
microscopy analysis (BacLight, duplicates).
[0065] FIG. 9. Biofilms grown for 3 months on uPVC coupons in AR
in-line with recycled water distribution system and spiked with S.
marcescens. Effect of exposure of the biofilms to 0, 100 nM and 500
nM SNP for 18 hours before a 10 minutes treatment with a range of
concentration of free chlorine: (A) viability counts on eutrophic
agar (LB.sub.10, triplicates); (B) percentage of surface coverage
using microscopy analysis (BacLight, duplicates).
[0066] FIG. 10. Effect of SNP on dispersal of Serratia marcescens
biofilms.
[0067] FIG. 11. Effect of SNAP on dispersal of Serratia marcescens
biofilms.
[0068] FIG. 12. Effect of SNP on dispersal of Vibrio cholerae
biofilms.
[0069] FIG. 13. Effect of SNAP on dispersal of Vibrio cholerae
biofilms.
[0070] FIG. 14. Enhancement by SNP treatment of the antimicrobial
activity of tetracycline (6 .mu.g/mL) on biofilms of Vibrio
cholerae.
[0071] FIG. 15. Effect of GSNO on dispersal of Vibrio cholerae
biofilms.
[0072] FIG. 16. Effect of SNP on dispersal of E. coil biofilms.
[0073] FIG. 17. Effect of SNP on dispersal of Bacillus
licheniformis biofilms.
[0074] FIG. 18. Effect of SNP on dispersal of Candida albicans
biofilms.
[0075] FIG. 19. Inhibition by SNP treatment of biofilm formation by
Staphylococcus epidermidis.
[0076] FIG. 20. Inhibition by SNP of attachment of Fusobacterium
nucleatum to a glass surface.
DETAILED DESCRIPTION OF THE INVENTION
[0077] P. aeruginosa is a ubiquitous soil- and water-borne
opportunistic pathogen that readily forms both single species and
multi-species biofilms. P. aeruginosa has also become a model
organism for studying biofilm formation and development. Recent
studies of P. aeruginosa biofilms have identified dispersal of
cells from the interior portions of microcolonies, and programmed
cell death of cells which can result in detachment and sloughing
events (Sauer et al., 2002, Pseudomonas aeruginosa displays
multiple phenotypes during development as a biofilm, J. Bact., 184:
1140-1154; Webb et al., 2003, Cell death in Pseudomonas aeruginosa
biofilm development, J. Bact., 185: 4585-4592). Subsequently,
programmed cell death has been reported in other model biofilm
forming bacteria (Mai-Prochnow et al., 2004, Biofilm development
and cell death in the marine bacterium Pseudoalteromonas tunicata,
Appl. Environ. Microbiol. 70: 3232-3238), in mixed species oral
biofilms (Hope et al., 2002, Determining the spatial distribution
of viable and non viable bacteria in hydrated microcosm dental
plaques by viability profiling, J. Appl. Microbiol, 1993: 448-455)
and in mixed species granular biofilms in waste water treatment
processes (Meyer at al., 2003, Microscale structure and function of
anaerobic-aerobic granules containing glycogen accumulating
organisms, FEMS Microbiol. Ecol., 45: 253-261), thereby suggesting
that programmed cell death is a general feature of bacterial
biofilm development. The exploitation, either by way of enhancement
or inhibition, of the mechanisms that trigger cell death and
detachment in biofilm cells will lead to novel technologies for the
manipulation of biofilms in a broad range of medical, industrial
and bioprocessing situations.
[0078] Utilising fluorescent dye-based systems for the detection
and analysis of reactive oxygen and nitrogen species (RONS) in
biofilms, the present inventors have found that the RONS
peroxynitrite (ONOO--) accumulates in P. aeruginosa biofilms.
[0079] Peroxynitrite is a potent oxidant with a wide range of
biological effects. It is capable of reacting with a number of
other biomolecules and causing cellular damage. An immediate
precursor of peroxynitrite is nitric oxide, a widespread
intercellular and intracellular signalling molecule in biological
systems. Nitric oxide rapidly reacts with a number of compounds,
including oxygen-derived radicals. In one such reaction nitric
oxide readily reacts with superoxide to yield the
peroxynitrite:
NO+O.sub.2-.fwdarw.ONOO--
[0080] The inventors have found that treatment of biofilms with low
non-toxic concentrations of a nitric oxide donor compound induces
programmed cell death and dispersal of cells from the biofilm,
leading to an increase in the ratio of planktonic to biofilm cells
and a reduction in surface coverage of the biofilm. Thus, disclosed
herein for the first time is evidence that low, non-toxic
concentrations of nitric oxide can be used to manipulate the
behavioural processes of biofilm cells. In contrast, higher, toxic
concentrations of nitric oxide do not achieve the dispersal of
cells from biofilms, but rather promote biofilm growth.
[0081] Accordingly, one aspect of the present invention relates to
a method for promoting dispersal of microorganisms from a biofilm,
the method comprising exposing the biofilm to an effective amount
of nitric oxide or at least one nitric oxide generating or
releasing agent. The invention also relates to methods for inducing
programmed cell death in a microorganism wherein the microorganism
is exposed to an effective amount of nitric oxide or at least one
nitric oxide generating or releasing agent, and to methods for
inhibiting biofilm formation and/or development wherein a surface
susceptible to biofilm formation is treated with an effective
amount of nitric oxide or at least one nitric oxide generating or
releasing agent.
[0082] The present inventors have also found that treatment with a
nitric oxide donor increases susceptibility of both biofilm and
planktonic cells to various antimicrobial agents. Accordingly, the
present invention also provides methods and compositions for
increasing the susceptibility of microorganisms to antimicrobial
agents.
[0083] The person skilled in the art will appreciate that nitric
oxide may be used directly to achieve the desired effect, or
alternatively any agent capable of generating or releasing nitric
oxide, may be used. The agent may generate or release nitric oxide
external to the organisms to be treated, or more typically in vivo.
Methods of the invention are exemplified herein using nitric oxide
donors such as sodium nitroprusside, but those skilled in the art
will appreciate that the invention is not so limited.
[0084] Examples of nitric oxide donors suitable for use in
accordance with the present invention include, but are not limited
to sodium nitroprusside (SNP), S-nitroso-L-glutathione (GSNO), GSNO
monoethyl ester, S-nitroso-N-acetylpenicillamine (SNAP),
glyco-SNAP, L-arginine,
N,N'-dinitroso-N,N'-dimethylphenylenediamine (BNN3),
N,N'-dinitrosophenylenediamine-N,N'-diacetic acid (BNN5), BNN5-Na,
BNN5 methyl ester, 2-hydroxybenzoic acid 3-nitrooxymethylphenyl
ester (B-NOD), dephostatin, 3,4-dephostatin, diethylamine NONOate,
diethylamine NONOate/AM, S,S'-dinitrosodithiol, S-nitrosocaptopril,
N.sup.G-hydroxy-L-arginine monoacetate salt, Angeli's salt,
1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene (NOC-5),
1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazene
(NOC-7),
6-(2-hydroxy-1-methyl-2-nitrisohydrazino)-N-methyl-1-hyxanamine
(NOC-9),
1-hydroxy-2-oxo-3-(N-ethyl-2-aminoethyl)-3-ethyl-1-triazene
(NOC-12), 2,2'-(hydroxynitrosohydrazono)bis-ethanamine (NOC-18),
(.+-.)-(E)-Methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide
(NOR-1),
(.+-.)-(E)-4-ethyl-2-[(E)-hydroxyiminol-5-nitro-3-hexenamide
(NOR-3), (.+-.)
-N-[(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridine
carboxamide (NOR-4), 4-phenyl-3-furoxancarbonitrile, PROLI/NO
(L-proline in methanolic sodium methoxide),
3-morphorlinosydnonimine (SIN-1), S-nitroso-N-valerylpenicillamine
(SNVP), spermine NONOate, ethyl nitrite and streptozotocin.
[0085] In general, nitric oxide donors, including S-nitroso,
O-nitroso, C-nitroso and N-nitroso compounds and nitro derivatives
thereof and metal NO complexes, but not excluding other NO
generating compounds, useful for the purposes of the present
invention may be found in "Methods in Nitric Oxide Research,"
edited by Feelisch, M., and Stamler, J. S., John Wiley & Sons,
New York, 1996, pages 71-115, the disclosure of which is
incorporated herein by reference. A range of additional nitric
oxide donors are known to those skilled in the art and the present
invention is not limited by the identity of the particular donor(s)
used. Indeed the selection of the appropriate donor for a
particular application of the invention may be made on a
case-by-case basis.
[0086] Nitric oxide generating or releasing agents are typically
used at low, non-toxic concentrations. The concentration may be
nanomolar, micromolar, or millimolar. In particular embodiments,
the concentration may be between about 1 nM and about 100 mM,
between about 10 nM and about 50 mM, between about 25 nM and about
50 mM, between about 50 nM and about 25 mM, between about 100 nM
and about 10 mM, between about 200 nM and about 1 mM, between about
1 nM and about 10mM, between about 10 nM and 50 .mu.M, between
about 10 nM and 25 .mu.M, between about 10 nM and 10 .mu.M, between
about 10 nM and 5 .mu.M, between about 10 nM and 1 .mu.M, or
between about 10 nM and 500 nM. The most suitable concentration to
achieve the desired effect will depend on a number of factors and
may be determined by those skilled in the art using routine
experimentation. Such factors include, but are not limited to, the
particular agent(s) used, the means or route of administration of
the agent(s), the nature, structure and age of the biofilm, the
species of organism to be treated and so on.
[0087] The present invention also provides compositions for
promoting dispersal of microorganisms from a biofilm, for inducing
programmed cell death in a microorganism, for inhibiting biofilm
formation and/or development and for increasing the susceptibility
of microorganisms to antimicrobial agents. Typically the
compositions provide means for carrying out the methods of the
invention.
[0088] The methods and compositions of the invention described
above find application in a wide range of environments and
circumstances. The following is a brief discussion of some general
areas of application of these methods and compositions. However
those skilled in the art will readily appreciate that any
environment or situation in which biofilm development is a problem
or in which it is desirable to inhibit microbial growth will be
potentially suitable for these methods and compositions.
[0089] One area of application of methods and compositions of the
invention is in marine, brackish water and freshwater anti-fouling
paints or coatings, for example in treating ship hulls, aquaculture
equipment, fishing nets or other in-water structures. The methods
and compositions also find application in a range of industrial and
domestic applications, including but not limited to water supply
reservoirs and feed pipes, drain pipes (domestic or industrial
scale), process equipment of, for example, cooling towers, water
treatment plants, dairy processing plants, food processing plants,
chemical manufacturing plants, pharmaceutical or biopharmaceutical
manufacturing plants, oil pipelines and oil refinery equipment, and
pulp and paper mills.
[0090] Compositions of the invention may also be used in coating
medical devices, including implantable medical devices, including
but not limited to venous catheters, urinary catheters, stents,
prostheses such as artificial joints, hearts, heart valves or other
organs, pacemakers, surgical plates and pins and contact lenses.
Other medical equipment may also be coated, such as catheters and
dialysis equipment. Methods and compositions of the invention also
find application in the management of infectious diseases. For
example, a variety of bacterial infections associated with biofilm
formation may be treated with methods and compositions of the
invention, such as cystic fibrosis, otitis media, bacterial
endocarditis, kidney stones, legionnaire's disease, urinary tract
infections, pulmonary infections, dental plaque, dental caries and
infections associated with surgical procedures or burns.
Accordingly, compositions of the invention may be formulated as
pharmaceutical compositions or form components of, for example,
surgical dressings, mouthwash, toothpaste or saline solutions.
[0091] Compositions according to the invention may be applied or
coated onto, or incorporated in the surface of an object/item of
interest well in advance of use of said object/item in, or exposure
of said object/item to an environment which comprises
biofilm-forming microorganisms, or a composition of the invention
may be applied or coated onto, or incorporated in the surface of an
object/item of interest immediately before use of that object/item
in, or exposure of said object/item to an environment which
comprises biofilm-forming microorganisms.
[0092] Compositions according to the invention may be in any
suitable form. For example a composition of the invention may be
formulated as a paint, wax, other coating, emulsion, solution, gel,
suspension, beads, powder, granules, pellets, flakes or spray. The
skilled addressee will recognise that the appropriate formulation
will depend on the particular application and the proposed route of
delivery.
[0093] Compositions of the invention typically also include
carriers, diluents or excipients. Suitable carriers, diluents and
excipients are known to those skilled in the art. The diluents,
adjuvants and excipients must be "acceptable" in terms of being
compatible with the other ingredients of the composition, and in
the case of pharmaceutical compositions, not deleterious to the
recipient thereof.
[0094] Carriers may be liquid or solid. In the case of liquid
carriers, the liquid may be an aqueous or non-aqueous solvent.
Typically for anti-fouling and other industrial applications, the
composition, for example in the form of a paint or other surface
coating, employs a carrier enabling the controlled release of the
active agent temporally and/or spatially. A variety of methods to
achieve controlled release of bioactive agents are known to those
skilled in the art and may include, for example, encapsulation of
the active agent in a suitable polymer or polymer-based product.
The polymer may be an organic or inorganic polymer, for example a
polyolefin, polyether, polyester, polyamide, polyurethane or
polypeptide. Suitable polymers for providing controlled release are
known to those skilled in the art, for example as disclosed in U.S.
Pat. No. 6,610,282, the disclosure of which is incorporated herein
by reference.
[0095] Typically, the rate of release of the substance is
determined by the properties of the polymer itself as well as
environmental factors (such as pH, temperature etc). Controlled
release systems are capable of delivering substances slowly and
continuously for up to several years. Those skilled in the art will
appreciate that a number of controlled release systems are
applicable to the delivery of agents according to the present
invention. By way of example only, release may be diffusion
controlled, chemically controlled or solvent activated.
[0096] In diffusion controlled systems, diffusion of the agent
trapped within a polymer matrix is the rate-determining factor for
the overall release rate. One type of diffusion controlled system
employs a reservoir device in which the agent forms a core
surrounded by an inert diffusion barrier. These systems include
membranes, capsules, microcapsules, liposomes, and hollow fibers.
Alternatively the device may be a monolithic device in which the
active agent is dispersed or dissolved in an inert polymer.
Diffusion through the polymer matrix is the rate-limiting step, and
release rates are determined in part by the choice of polymer and
its consequent effect on the diffusion and partition coefficient of
the agent to be released.
[0097] In typical chemically controlled systems a polymer degrades
over time and releases an agent in an amount proportional to the
gradual erosion. Chemical control can be achieved using bioerodible
or pendant chains. In a bioerodible system the agent is ideally
distributed uniformly throughout a polymer in the same way as in
monolithic diffusion systems. As the polymer surrounding the agent
is eroded, the agent escapes. In a pendant chain system, the agent
may be covalently bound to the polymer by a chemistry allowing for
release by any desired and practicable physical or chemical means
known in the art such as by, for example, bond scission owing to
water or enzymes.
[0098] In typical solvent-activated controlled systems, the active
agent (which may be nitric oxide, a nitric oxide generating or
releasing agent, or a nitric oxide scavenger or an antioxidant) is
dissolved or dispersed within a polymeric matrix and is not able to
diffuse through that matrix. Osmotic pressure is used as the
driving force for release of the agent. In one type of
solvent-controlled system, as the environmental fluid (e.g., water)
penetrates the matrix, the polymer (e.g. a hydrogel) swells and its
glass transition temperature is lowered below the environmental
(host) temperature. Thus, the swollen polymer is in a rubbery state
and allows the, active agent contained within to diffuse through
the encapsulant.
[0099] Chemical bonding of a bioactive agent to a polymer can be
accomplished in several general ways based on different methods of
synthesis well known to those skilled in the art including:
reaction on preformed polymers; reactions on naturally-occurring
polymers; polymerization of vinyl monomers containing the active
ingredient; and step growth polymerizations. When the bioactive
agent is chemically bonded to a polymer, the bond has to be cleaved
by a chemical reaction-typically enzymatic, hydrolytic, thermal, or
photochemical. A variety of chemical and physical variables can
affect the rate of bond cleavage and subsequent release of
chemically attached materials from polymers including the nature of
the labile bond, length of the spacer group, molecular weight,
hydrophilicity, neighbouring group effects, environmental factors
and physical form and dimensions.
[0100] In anti-fouling applications, self-polishing antifouling
coatings are known in the art. Such coatings are typically based on
polymers of tributyltin methacrylate, methyl methacrylate, and film
softening monomers such as 2-ethylhexyl acrylate. An organotin
polymer typically acts as the paint binder. Such paints may also
contain a toxicant additive such as cuprous oxide or a triorganotin
compound. In addition, the usual paint additives such as pigments,
thixotropic agents may also be present. In normally alkaline
seawater, the polymeric organotin binder is gradually hydrolyzed,
and the tributyltin is liberated in a form that is an active
antifoulant. The hydrolyzed polymer formed is water-soluble or
water-swellable and is easily eroded off the surface by moving
seawater, exposing a fresh surface of paint.
[0101] For pharmaceutical applications, a number of suitable
controlled release systems are known in the art. For example,
polymeric colloidal particles or microencapsulates (microparticles,
microspheres or nanoparticles) in the form of reservoir and matrix
devices may be employed, or the agent may be contained by a polymer
containing a hydrophilic and/or leachable additive eg, a second
polymer, surfactant or plasticiser, etc. to give a porous device,
or a device in which the drug release may be osmotically
`controlled` (both reservoir and matrix devices). Large cage-like
molecules such as the C60 Buckminster-fullerenes ('Buckyballs') or
hyperbranched (starburst) dendrimers may also be used.
[0102] Those skilled in the art will readily appreciate that the
delivery systems and methods described above are merely examples of
suitable methods and systems that may be employed in the present
invention. Any other suitable carriers and delivery systems may be
employed to achieve the desired means of application of agents
according to embodiments of the present invention.
[0103] Examples of pharmaceutically acceptable diluents are
demineralised or distilled water; saline solution; vegetable based
oils such as peanut oil, safflower oil, olive oil, cottonseed oil,
maize oil, sesame oils such as peanut oil, safflower oil, olive
oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut
oil; silicone oils, including polysiloxanes, such as methyl
polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane;
volatile silicones; mineral oils such as liquid paraffin, soft
paraffin or squalane; cellulose derivatives such as methyl
cellulose, ethyl cellulose, carboxymethylcellulose, sodium
carboxymethylcellulose or hydroxypropylmethylcellulose; lower
alkanols, for example ethanol or iso-propanol; lower aralkanols;
lower polyalkylene glycols or lower alkylene glycols, for example
polyethylene glycol, polypropylene glycol, ethylene glycol,
propylene glycol, 1,3-butylene glycol or glycerin; fatty acid
esters such as isopropyl palmitate, isopropyl myristate or ethyl
oleate; polyvinylpyrrolidone; agar; carrageenan; gum tragacanth or
gum acacia, and petroleum jelly. Typically, the carrier or carriers
will form from 1% to 99.9% by weight of the compositions.
[0104] For pharmaceutical applications, compositions may be
formulated for delivery by any route, for example oral, topical,
intracavitary, intravesical, intramuscular, intraarterial,
intravenous, intranasal, intrapulmonary or subcutaneous.
[0105] As the inventors have discovered that nitric oxide and
nitric oxide generating or releasing agents result in increased
sensitivity of microorganisms to antimicrobial agents, methods of
the present invention may be employed in combination with at least
one antimicrobial agent. Any suitable antimicrobial agents may be
used, for example antibiotics, detergents, surfactants, agents that
induce oxidative stress, bacteriocins and antimicrobial enzymes,
peptides and phage. The antimicrobial agents may be natural or
synthetic. Indeed the antimicrobial agent employed may be selected
for the particular application of the invention on a case-by-case
basis, and those skilled in the art will appreciate that the scope
of the present invention is not limited by the nature or identity
of the particular antimicrobial agent. By way of example only,
suitable antibiotics include but are not limited to .beta.-lactams,
monopenems, carboxypenems, aminoglycosides, quinolones, macrolides,
lincozamides, tetracyclines, streptogramins, glycopeptides,
rifamicins, sulphonamides chloramphenicol, nalidixic acid,
azole-containing compounds and peptide antibiotics. Antimicrobial
enzymes include but are not limited to lipases, pronases, lyases
(e.g. alginate lyases) and various other proteolytic enzymes and
nucleases.
[0106] It will be readily appreciated by those skilled in the art
that according to the methods of the invention each component of
the combination may be administered at the same time, or
sequentially in any order, or at different times, so as to provide
the desired effect. Alternatively, the components may be formulated
together in a single dosage unit as a combination product.
Accordingly, compositions of the invention may comprise, in
addition to nitric oxide and/or at least one nitric oxide
generating or releasing agent, at least one antimicrobial
agent.
[0107] As described herein, the present inventors have also found
that use of a nitric oxide scavenger reverses the effect observed
with the nitric oxide donor SNP on biofilm and planktonic growth,
thereby providing avenues for the inhibition of programmed cell
death and inhibition of cell dispersal from biofilms and the
maintenance and/or enhancement of activity of biofilms in
circumstances where this is beneficial.
[0108] Accordingly, aspects of the present invention relate to
methods for maintaining and/or enhancing the functioning of a
biofilm, wherein the biofilm is exposed to at least one nitric
oxide scavenger and/or at least one antioxidant. The invention also
relates to methods for inhibiting programmed cell death in a
microorganism, wherein the biofilm is exposed to at least one
nitric oxide scavenger and/or at least one antioxidant. The present
invention further provides compositions for carrying out the above
methods.
[0109] Thus, methods and compositions of the invention enable the
manipulation of biofilm longevity, viability, density, activity
and/or efficacy via the use of nitric oxide scavenging molecules
and RONS-quenching molecules. Agents suitable for maintaining
and/or enhancing the functioning of a biofilm or inhibiting
programmed cell death include nitric oxide scavengers such as
2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide (PTIO),
carboxy-PTIO, N-(dithiocarboxy)sarcosine (DTCS),
N-methyl-D-glucamine dithiocarbamate (MGD), (+)-rutin hydrate and
haemoglobin, and antioxidants such as thioredoxin, superoxide
dismutase, glutathione and ascorbic acid. Those skilled in the art
will readily appreciate that a number of other nitric oxide
scavengers and antioxidants known in the art are equally applicable
to the present invention, and the present invention is not limited
by the identity of the particular agents used. Indeed the selection
of the appropriate agent for a particular application of the
invention may be made on a case-by-case basis. Compositions
containing such agents may be formulated as described above.
[0110] Those skilled in the art will appreciate that the methods
and compositions of the present invention relating to biofilm
regulation are applicable to single species or mixed species
biofilms. Bacterial species to which the present invention relates
may be any species capable of forming a biofilm or contributing to
a biofilm. Targeted microbial species may include fungi, including
yeasts and filamentous fungi, and Gram positive and Gram negative
bacteria. Biofilms of interest to the present invention may
comprise microorganisms selected from, but not limited to Candida
spp. (including C. albicans), Hormoconis spp. (including H.
resinae), Pseudomonas spp. such as P. aeruginosa, Pseudoalteromonas
spp. such as P. tunicata, Staphylococcus spp. such as S. aureus
(including methicillin-resistant and vancomycin-resistant S.
aureus) and S. epidermidis, Streptococcus spp. such as S. mutansl
S. sobrinus, Shigella spp. such as S. flexeri, S. dysenteria,
Mycobacterium spp. such as M. tuberculosis, Enterococcus spp. such
as E. faecalis, Escherichia spp. such as E. coli, Salmonella spp.
such as S. typhimurium, S. typhi and S. enteritidis, Legionella
spp. such as L. pneumophila, Haemophilus spp. such as H.
influenzae, Bacillus spp. such as B. Licheniformis,
sulfate-reducing and iron-reducing bacteria (such as Desulfovibrio
spp., including D. vulgaris and D. desulfuricans, Shewanella spp.,
including S. putrefaciens, Geobacter spp., including G.
metallireducens), Klebsiella spp. such as K. pneumoniae, Proteus
spp. such as P. mirabilis, Aeromonas spp., Arthrobacter spp.,
Micrococcus spp., Serratia spp. such as S. marcescens,
Porphyromonas spp. such as P. gingivalis, Fusobacterium spp., such
as F. nucleatum and Vibrio spp, such as V. cholerae. The microbial
species may be aerobic, anaerobic, facultative, aerotolerant,
aerophobic, or microaerophilic. Alternatively those skilled in the
art will appreciate that in some applications of the present
invention, the identities of the particular species in the mixed
communities of the biofilm to be treated are undetermined and are
not critical to the applicability of the invention.
[0111] The present invention will now be further described in
greater detail by reference to the following specific examples,
which should not be construed as in anyway limiting the scope of
the invention.
EXAMPLES
[0112] For the biofilm studies described in Example 1 to 3,
Pseudomonas aeruginosa strains PAO1 and PAO1-GFP, containing a
chromosomal insertion of green fluorescent protein (GFP),
generously donated by Marie Allesen-Holm, were used, Overnight
cultures were routinely performed in Luria Bertani (LB) medium,
with shaking at 37.degree. C. Biofilm and planktonic experiments
were conducted in modified M9 minimal medium as described elsewhere
(Webb et al., 2003, Cell death in Pseudomonas aeruginosa biofilm
development, J. Bacteriol., 185: 4585-4592) with 5 mM glucose for
continuous culture experiments and 20 mM for batch experiments.
Example 1
Cell Death and Dispersal in Biofilms Correlate with the
Accumulation of Peroxynitrite in Mature Microcolonies
[0113] P. aeruginosa PAO1 were grown in continuous culture flow
cells (channel dimensions, 1.times.4.times.40 mm) at room
temperature as previously described (Moller et al., 1998, In situ
gene expression in mixed-culture biofilms: evidence of metabolic
interactions between community members, Appl. Environ. Microbiol.,
64: 721-732) for biofilm generation. Channels were inoculated with
0.5 mL of overnight cell cultures and incubated without flow for 1
hour at room temperature. Flow was then started with a mean flow
velocity in the flow cells of 0.2 mm.s.sup.-1, corresponding to
laminar flow with a Reynolds number of 0.02.
[0114] To investigate cell death during biofilm development,
biofilms were stained with the LIVE/DEAD BacLight bacterial
viability kit (Molecular Probes) in which SYTO9 is used to
specifically stain live cells and propidium iodide is used to
specifically stain dead cells. Stock solutions of the stains were
diluted to 3 .mu.L.mL.sup.-1 in modified M9 medium and injected
into the flow channels. Live SYTO 9-stained cells and dead
propidium iodide-stained cells were visualized with a confocal
laser scanning microscope (Olympus) with fluorescein isothiocyanate
and tetramethyl rhodamine isocyanate optical filters, respectively.
It was observed that after 7 days, biofilms of P. aeruginosa
undergo highly reproducible patterns of cell death and dispersal,
and that these events lead to the formation of hollow colonies
within biofilms (FIG. 1A--white arrow points to hollow structure;
black arrow points to dead cells).
[0115] To investigate the role of specific reactive and nitrogen
species (RONS) and detect specific RONS that accumulate in biofilms
structures during death and dispersal, a series of reactive
fluorescent dyes, each targeting a different RONS, were injected in
the flow channels and were incubated 30 minutes in the dark before
confocal laser scanning microscopy. The RONS investigated were
nitric oxide (NO), peroxynitrite (ONOO--), hydrogen peroxide
(H.sub.2O.sub.2) and superoxide radicals (O.sub.2-.cndot.).
DAFFM-DA (Molecular Probes), 5 mM stock in DMSO, was used at 100
.mu.M for the detection of nitric oxide (Kojima et al., 1999,
Fluorescent indicators for imaging nitric oxide production,
Angew.Chem. Int. Ed. Engl. 38: 3209-3212), Dihydrorhodamine 123
(DHR) (Sigma), stock 2.5 mg.mL.sup.-1 in ethanol, was used at 15
.mu.M for the detection of peroxynitrite (Crow, 1997,
Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive
indicators of peroxynitrite in vitro: implications for
intracellular measurement of reactive nitrogen and oxygen species,
Nitric Oxide, 1: 145-157), carboxy-H.sub.2DCF-DA (Molecular
Probes), 10 mM stock in DMSO, was used at 100 .mu.M for the
detection of hydrogen peroxide, and hydroethidine (HEt) (Sigma), 1
mg.mL.sup.-1 stock in 1% DMSO in Phosphate Buffered Saline (PBS),
was used at 10 .mu.M for the detection of superoxide radicals
(Bindokas et al., 1996, Superoxide production in rat hippocampal
neurons: selective imaging with hydroethidine, J. Neurosci. 16:
1324-1336). Stock solutions were kept frozen and covered from
light. Final solutions were freshly made in modified M9 medium
before use.
[0116] As shown in FIG. 1B, the bacteria in the biofilm showed a
low level of autofluorescence, revealed by the control images.
Positive fluorescence was detected with two of the RONS-specific
dyes: HEt detecting superoxide radicals, O.sub.2-.cndot. and, at
considerably higher level of fluorescence, DHR detecting
peroxynitrite, ONOO--. The light field images (left panel) revealed
that the fluorescence occurred in mature microcolonies that had
undergone death and dispersal events as shown in FIG. 1A. The
negative results obtained with H.sub.2DCF, for the detection of
hydrogen peroxide, correlate with the overexpression of catalase
previously reported in P. aeruginosa biofilms (Stewart et al.,
2000, Effect of catalase on hydrogen peroxide penetration into
Pseudomonas aeruginosa biofilms, Appl. Environ. Microbiol. 66:
836-838). DHR is specific to peroxynitrite and cannot be oxidized
by other RONS alone (Crow at al., 1999). Because peroxynitrite is
the direct product of superoxide and nitric oxide, it was
surprising not to detect nitric oxide with DAFFM. However, nitric
oxide is extremely reactive, and the reaction between nitric oxide
and superoxide is known to occur instantaneously, in a
diffusion-limited manner (Kelm at al., 1997, The nitric
oxide/superoxide assay. Insights into the biological chemistry of
the NO/O.sub.2- interaction, J. Biol. Chem. 272: 9922-9932), which
may prohibit the detection of nitric oxide with DAFFM.
[0117] The results presented above demonstrate that the RONS
peroxynitrite accumulates in biofilm cells at high cell densities
and triggers cell death in microcolonies within mature
biofilms.
Example 2
Biofilm Versus Planktonic Growth in P. aeruginosa is Regulated by
Nitric Oxide
[0118] Nitric oxide is a widespread intercellular and intracellular
signalling molecule in biological systems. It is also the key
precursor of peroxynitrite, a potent oxidant with a wide range of
biological effects. Nitric oxide readily reacts with superoxide to
yield peroxynitrite:
NO+O.sub.2-.fwdarw.ONOO
[0119] The inventors therefore investigated the effect of the
nitric oxide donor, sodium nitroprusside (SNP), on planktonic and
biofilm growth of P. aeruginosa. SNP releases nitric oxide in vivo
(Smith et al., 2001, Mechanisms of nitric oxide release from
nitrovasodilators in aqueous solution: reaction of the
nitroprusside ion ([Fe(CN).sub.5NO].sub.2-) with L-ascorbic acid, J
Inorg Biochem, 87: 165-173).
[0120] Biofilms in 96-well plates were used for these experiments.
100 .mu.L of 1/1000 overnight cultures of PAO1-GFP diluted in
modified M9 medium were inoculated in 96-well plates (Sarstedt) and
grown for 24 hours, at 37.degree. C. with 120 rpm shaking. SNP was
added to the cultures at concentrations of between 25 nM and 100
mM. 4 replicates per treatment were used. After overnight growth,
the supernatant was transferred to wells of a new plate. The wells
were washed twice with PBS and stained for 20 minutes with 120
.mu.L of crystal violet. The wells were then washed again three
times with PBS and diluted in 120 .mu.L of absolute ethanol.
Biofilm formation was quantified by measurement of OD.sub.490nm.
and planktonic growth was quantified by fluorescence
measurement.
[0121] As shown in FIG. 2, at high concentrations (in the
millimolar range) an increase in biofilm formation and a decrease
in planktonic growth was observed (25 mM-100 mM SNP) in comparison
to untreated biofilm. At these concentrations, SNP is toxic to
various bacterial species; the SNP releases high, toxic
concentrations of nitric oxide (Joannou et al., 1998,
Characterization of the bactericidal effects of sodium
nitroprusside and other pentacyanonitrosyl complexes on the food
spoilage bacterium Clostridium sporogenes, Appl Environ Microbiol,
64: 3195-3201; Kelley et al., 1998, Inducible nitric oxide synthase
expression is reduced in cystic fibrosis murine and human airway
epithelial cells, J Clin Invest, 102: 1200-1207). At lower
concentrations, in the micromolar and nanomolar ranges, a decrease
in biofilm formation and an increase in planktonic growth was
observed. The highest effect was repeatedly observed with 500 nM
SNP and this concentration was used for subsequent experiments
described in this Example and Example 3.
[0122] To confirm the role of nitric oxide in the observed events,
two different nitric oxide donors, S-nitroso-L-glutathione (GSNO)
and S-nitroso-N-acetylpenicillamine (SNAP) were used in place of
SNP, each at concentrations of 1 .mu.M. As illustrated in FIG. 3,
similar to SNP, both of these nitric oxide donors also produced
decreases in biofilm formation and increases in planktonic growth,
although to a lesser extent than was observed with SNP.
[0123] These results suggest that at low concentrations, nitric
oxide signals a transition from biofilm to planktonic
phenotype.
[0124] FIG. 3 also shows the effect of the nitric oxide scavenger
2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide (PTIO).
PTIO was added to a growing biofilm at 1 mM concentration in
addition to SNP at 500 nM. The addition of PTIO reduced the SNP
effect by 40% or more in both planktonic and biofilm
phenotypes.
Example 3
Exposure to Low Levels of Nitric Oxide Increases the Sensitivity of
P. aeruginosa Cells to Antimicrobial Agents
[0125] Planktonic cells are known to be up to 1000 times more
sensitive towards antibiotics than biofilm cells (Brooun et al.,
2000, A dose-response study of antibiotic resistance in Pseudomonas
aeruginosa biofilms, Antimicrob Agents Chemother, 44: 640-646;
Davies, 2003, Understanding biofilm resistance to antibacterial
agents, Nat Rev Drug Discov, 2: 114-122). One of the major
difficulties in combating mature biofilms is this reduced
sensitivity to antimicrobial agents. The results described above in
Example 2 demonstrate that nitric oxide promotes the planktonic
mode of growth over the more resistant biofilm phenotype. The
inventors therefore investigated whether nitric oxide exposure may
also restore antimicrobial sensitivity to biofilm cells. The
effects of various antimicrobial agents on P. aeruginosa biofilms
and planktonic cells that were exposed to low levels of nitric
oxide were examined.
[0126] To test the sensitivity of cells, a broad range of
antimicrobial compounds were examined: the antibiotic tobramycin
(Sigma) that irreversibly inhibits bacterial protein synthesis,
used at a final concentration of 100 .mu.M; the surfactant sodium
dodecyl sulphate (SDS) used at 0.1%; and the oxidative stress
inducing agents hydrogen peroxide (H.sub.2O.sub.2; at 10 mM
concentration) and hypochlorous acid (HOCl; at 8 mM
concentration).
[0127] Biofilms were grown in petri dishes containing microscope
glass slides (76.times.26 mm, Superfrost, Menzel Glaser). To
prevent contamination, the slides were autoclaved and the petri
dishes sterilised by 30 minutes exposure to ultraviolet. 25 mL of
1/1000 overnight cultures of PAO1-GFP diluted in modified M9 medium
were inoculated in the plates and grown for 24 hours, at 37.degree.
C. with 50 rpm shaking, allowing biofilm formation on the slides.
OD.sub.600nm readings of the supernatant were determined. The
slides were then rinsed in sterile PBS. For the antimicrobial
sensitivity assays, the slides were incubated with modified M9
medium alone (negative control), or with 500 .mu.L of the
antimicrobial agent diluted in modified M9 medium, for 30 minutes
in a humid chamber and rinsed again in PBS. The biofilms on the
slides were stained with 250 .mu.L of LIVE/DEAD staining as
described above for 20 minutes in a humid chamber. Seven confocal
pictures per slide were randomly taken and the percentage of
surface coverage was quantified with image analysis.
[0128] A dramatic reduction, up to 95%, in the number of biofilm
cells after the combination treatments (SNP and an antimicrobal
agent) was reproducibly observed (FIGS. 4A and 4B). The highest
effect was obtained with tobramycin, an antibiotic commonly used to
treat cystic fibrosis patients.
[0129] The ability of SNP to increase the sensitivity of
pre-established P. aeruginosa biofilms was also investigated, as
illustrated in FIG. 5. Biofilms were grown as described above, with
the exception that the initial 24 hours of development was in the
absence of SNP. After 24 hours, the media was changed and replaced
with media containing 500 mM SNP (in treatment biofilms; SNP absent
in controls). Biofilms were grown for a further 24 hours prior to
the addition of either Tb at 100 .mu.M or H.sub.2O.sub.2 at 10 mM
for 30 minutes or exposure to UV light (19 W, 254 nm for 30 min, 30
cm from the lamp). Sensitivity was determined as described above
with respect to FIG. 4. As shown in FIG. 5, a dramatic reduction in
biofilm surface coverage and the number of biofilm cells was
observed following combination treatment of pre-established
biofilms.
[0130] To investigate the effect of combination treatment of SNP
and an antimicrobial agent on planktonic cells, overnight cultures
of PAO1-GFP were diluted 1/1000 in modified M9 medium containing 20
mM of glucose with or without 500 nM SNP. After 24 hours the cells
were diluted 1/10 in the antimicrobial solution and incubated 2
hours at room temperature. CFU plate counts were performed to
assess the viability of the bacteria. The SNP pre-treatment on
planktonic cells resulted in a further 2 log decrease in CFU counts
after H.sub.2O.sub.2 and tobramycin exposures when compared to
untreated P. aeruginosa cells (FIG. 6). Interestingly, although
treatment with 500 nM SNP caused an increase in the optical density
of planktonic cultures compared to untreated controls (FIG. 3), an
equivalent increase in CFU counts (viability) was not observed
following exposure to tobramycin or hydrogen peroxide with SNP
pre-treatment.
[0131] Without wishing to be bound by theory, the inventors suggest
the results described above indicate that nitric oxide induces a
planktonic "dispersal" physiology in P. aeruginosa biofilms and
therefore enhances their sensitivity to antimicrobial agents.
Example 4
Nitric Oxide Induces Dispersal of Cells in Floc Biofilms
[0132] In view of the results described in Example 2 for
surface-associated biofilms, the ability of nitric oxide to induce
dispersal from a non surface-associated mixed species biofilm was
investigated.
[0133] Four parallel bioreactors (30 mL flasks) were operated under
acetate-fed denitrifying conditions (anoxic) for 13 days in batch
feeding mode (1 decant and feed cycle/day). The aim of these
experiments was to demonstrate that externally-supplemented NO,
generated by SNP can influence sludge flocculation, cell death and
dispersal in an activated sludge floc (biofilm) system.
[0134] The bioreactors were seeded with activated sludge collected
from St Marys Sewage Treatment Plant (STP) (Sydney, NSW, Australia)
on 18 Nov. 2004. The bioreactors were operated under anoxic
conditions at room temperature. Anoxic conditions were maintained
by the flushing/sparging of N.sub.2 gas into the sludge after
feeding with a mixture of 3 mL acetate medium and 12 mL nitrite
medium, in addition to 1 mL of SNP/water to achieve the desired
concentrations. The bioreactors were operated on a 24 hour cycle,
which consisted of 23.5 hour of anoxic reaction followed by a 10
minute settling and decant of 15 mL of supernatant.
[0135] The medium for the bioreactors was made up of two
components: a carbon medium base; and a nitrogen medium base. For
each cycle, the medium consisted of 1 volume of the carbon medium
base and 4 volumes of the nitrogen medium base. The carbon medium
base comprised (per litre) 6.587 g CH.sub.3COONa, 0.042 g
CaCl.sub.22H.sub.2O, 0.090 g MgSO.sub.4.7H.sub.2O, 0.160 g
MgCl.sub.2.6H.sub.2O, 0.011 g KH.sub.2PO.sub.4, 0.026 g
Na.sub.2HPO.sub.4.12H.sub.2O, 0.122 g Bacto peptone (Difco
Laboratories, USA), 0.020 g Bacto yeast extract (Difco
Laboratories), 0.025 g NH.sub.4Cl, and 0.3 ml nutrient solution as
previously described (Bond et al., 1999, Identification of some of
the major groups of bacteria in efficient and nonefficient
biological phosphorus removal activated sludge systems, Appl
Environ Microbiol, 65: 4077-84). The medium was made up with
Milli-Q water and sterilised by autoclaving.
[0136] The nitrogen medium base comprised (per litre) 8.972 g
NO.sub.2Na and was made up with reverse-osmosis-deionised water.
The acetate:NO.sub.2---N ratio in the combined medium was
maintained at 2.73:1.
[0137] To investigate the role of specific reactive and nitrogen
species (RONS) and to detect specific RONS that accumulate in
biofilm structures during death and dispersal, a series of reactive
fluorescent dyes, each targeting a different RONS (at
concentrations outlined in Example 1), were combined with 500 mL of
activated sludge taken from St Marys STP and were incubated for 30
minutes in the dark before confocal laser scanning microscopy. The
RONS investigated were hydrogen peroxide (H.sub.2O.sub.2), nitric
oxide (NO), peroxynitrite (ONOO--) as well as superoxide
(O.sub.2-.cndot.) and hydroxyl (OH) radicals. A control sample
(with no fluorescent RONS dye present) was used to establish CLSM
image collection levels for subsequent fluorescent probes. As shown
in Table 1, positive fluorescence was detected with two of the
RONS-specific dyes: intracellular hydrogen peroxide,
H.sub.2O.sub.2- and, at higher levels of fluorescence, DHR123
detecting peroxynitrite, ONOO--.
TABLE-US-00001 TABLE 1 Assessment of RONS present in St Marys STP
(seed) sludge. Fluorescent probe RONS Observation Control -- -
Amplex Red .sub.Extracellular H.sub.2O.sub.2 + DCF
.sub.Intracellular H.sub.2O.sub.2 ++ DHR123 ONOO.sup.- +++ HEt
O.sub.2.sub.-.cndot. - DAF-FM NO + TEMPO-9 HO/O.sub.2.sub.- -
[0138] To investigate and observe sludge flocs and planktonic cells
during biofilm development, biofilms were stained with the
LIVE/DEAD BacLight bacterial viability kit (Molecular Probes).
Stock solutions of the stains were diluted to 1 .mu.L mL.sup.-1 in
1.5 mL microcentrifuge tubes containing 500 mL of activated sludge
samples taken from each bioreactor. Samples (20 .mu.L) were mounted
onto glass slides, where cells were visualized with a confocal
laser scanning microscope (Olympus). Observations from each
bioreactor on day 13 were made for numbers of flocs and planktonic
cells. As shown in FIG. 7, substantial dispersal of cells from the
treated floc biofilm was observed (FIG. 7B) in comparison to the
untreated biofilm (FIG. 7A, Table 2.) after 13 days of SNP
treatment.
TABLE-US-00002 TABLE 2 Assessment of SNP effect on various sludge
parameters after 13 days incubation under anoxic conditions. [SNP]
Planktonic cells 0 (control) + 1 .mu.M ++ 100 .mu.M ++ 10 mM +++ (+
few planktonic cells, <5 cells/field of view; +++ abundant
dispersal cells, uncountable, >500 cells/field of view).
[0139] The greatest levels of dispersal and floc disruption were
observed using an SNP concentration of 10 mM. However, due to the
large size of the flocs (in many cases >200 .mu.m) the actual
concentration of NO presented to cells inside flocs is likely to be
considerably lower. Moreover, the release of NO from the NO donors
is dependant on complex chemical reactions and the effective
concentration of NO released can be up to 1-2 log lower than the
SNP concentrations used (Smith and Dasgupta, 2001, Mechanisms of
nitric oxide release from nitrovasodilators in aqueous solution:
reaction of the nitroprusside ion ([Fe(CN)5NO]2-) with L-ascorbic
acid, J. Inorg. Biochem. 87: 165-173).
Example 5
Nitric Oxide Induces Dispersal of Cells in Mixed Species
Biofilms
[0140] 5.1 Materials and Methods
[0141] 5.1.1 Model Distribution System
[0142] The biofilm sampling site (BSS) described by Storey and Ash
bolt (Storey M. V. and Ashbolt N. J. (2001), "Persistence of two
model enteric viruses (B40-8 and MS-2 bacteriophages) in water
distribution pipe biofilms", Water Sci Technol. 43 (12): 133-8) was
used in this study. Model potable and recycled water system
biofilms were grown in 2 continuous flow, annular reactors (model
920, BioSurfaces Technologies, Bozeman, Mont.) at the BSS. An
annular reactor (AR) consists of a rotating polycarbonate inner
cylinder and a stationary glass outer cylinder, separated by a
water-filled annular cavity. Sixty stainless steel (SS) and
unplasticized polyvinyl chloride (uPVC) coupons (15 mm.times.40 mm
available surface area) were placed on the exposed face of the
inner-rotating cylinder of the ARs, which received potable and
recycled water respectively, at a rate of 30 L.h.sup.-1 making the
hydraulic retention time 2.2 minutes. The annulus rotation speed
was set to provide a linear velocity similar to the mean hydraulic
demand of the distribution pipe (0.32 L.s.sup.-1). Biofilm coupons
were sterilized in 1 g.L.sup.-1 sodium hypochlorite for 2 hours and
rinsed with sterile Milli-Q water prior to being placed in each
experimental device. Biofilms were allowed to grow on coupon
surfaces for a period of 90 days. After this time the inlet flows
were stopped and the ARs were enriched with tetracycline and
ampicillin resistant Serratia marcescens at a final concentration
of approximately 10.sup.7 CFU.mL.sup.-1. S. marcescens cells were
allowed to settle in the biofilms on the SS and uPVC coupon
surfaces for 2 weeks, Unsettled cells were removed from the system
by reconnecting the potable and recycled water inlet flows for one
week. S. marcescens had previously been harvested from selective LB
agar plates (supplemented with 50 .mu.g.mL.sup.-1 tetracycline and
100 .mu.g.mL.sup.-1 ampicillin) after 24 hours incubation at
30.degree. C. and washed three times in phosphate buffered saline
(PBS).
[0143] 5.1.2 Experimental Design
[0144] Coupons were carefully removed from each biofilm device
using sterile forceps. SS coupons (potable) and uPVC coupons
(recycled) were removed from each experimental device and
transferred to bioreactors in the laboratory for NO exposure. The
bioreactors consisted of 1 L polypropylene (PP) beakers with bottom
inlet and top outlet covered with aluminium foil and containing PP
microscope slide racks (Kartell, Italy) that were modified to fit
the coupons. The racks were placed 2 cm above the bottom on a PP
stand, and a circular flow was generated with magnetic stirring to
simulate the hydraulic shear stress at the wall of a pipe. The
bioreactors were sterilized with 1 g.L.sup.-1 sodium hypochlorite
for 2 hours and rinsed with sterile Milli-Q water prior to
receiving coupons harboring the biofilms. For each type of biofilm
established from potable or recycled water systems, the coupons
were randomly placed in 3 separate bioreactors where they were
exposed for 18 hours to a continuous flow of autoclaved potable
water (sterile and dechlorinated), pH 7.8, containing 0, 100 nM or
500 nM of the NO donor sodium nitroprusside (SNP), at a flow rate
of 50 mL h.sup.-1.
[0145] The coupons were then carefully transferred into 25 mL glass
vials containing 20 mL of conventional chlorine treatments. A range
of chlorine treatments were freshly prepared by diluting 2.4 M
hypochlorite solution in 1/4 strength Ringers solution and
calibrated using a Pocket Colorimeter II (HACH, Loveland, Colo.),
After 10 minutes of gentle shaking (75 rpm), the reactions were
stopped by adding sodium thiosulfate at a final concentration of
100 .mu.M. For each NO/chlorine combinatorial treatment, triplicate
coupons were used for viability counts and duplicate coupons for
microscopy analysis.
[0146] 5.1.3 Analytical Methods
[0147] The coupons were processed for viability counts and
microscopy analysis.
[0148] LIVE/DEAD.RTM. BacLight Bacterial Viability Kit (Molecular
Probes, Oreg., USA) was used to stain cells in the biofilms. Two
stock solutions of stain (SYTO 9 and propidium iodide) were each
diluted to a concentration of 3 .mu.L.mL.sup.-1 in 1/4 strength
Ringers solution and the coupons were stained with 150 .mu.L of the
staining mix and covered with a thin coverslip (10.5.times.35 mm,
ProSciTech, Kirwan, Australia). The coupons were observed under
epifluorescence microscopy (Leica model DMR), and biofilm cells
were enumerated using an image analysis system (ImageJ, NIH).
[0149] For viability counts, the coupons were placed in sterile
stomacher bags (101.times.152 mm) (Seward, UK) containing 25 mL 1/4
strength Ringers solution with 100 .mu.M sodium thiosulfate. Bags
were heat-sealed and hand rubbed to initiate the disaggregation of
attached biofilms. The coupons were sonicated at 400 W (Branson
2210 Sonicator) for 60 s, then stomached for 60 s (Seward
Stomacher.RTM. 80, Seward, UK) to remove and homogenize remaining
biofilm. The homogenate was then aseptically removed from stomacher
bags. Heterotrophic plate counts (HPC) were performed using a
pour-plate technique on oligotrophic R2A agar (Oxoid, England), and
eutrophic Luria Bertani (LB) agar plates. The plates were incubated
at 30.degree. C. and counts were carried out after 7 days. S.
marcescens colonies were morphologically identified and confirmed
by plating the colonies on selective LB agar supplemented with 50
.mu.g.mL.sup.-1 tetracycline and 100 .mu.g.mL.sup.-1 ampicillin.
Sonication and stomaching techniques were previously examined for
their efficacy on biofilm removal through the recovery of HPCs.
[0150] Analysis of variance (ANOVA) tests at a significance level
of 95% were used to compare the impact of the various combinations
of low doses of NO, and chlorine disinfectant on biofilm
growth.
[0151] 5.2 Results
[0152] 5.2.1 Potable Water Biofilms Spiked with Serratia
marcescens
[0153] The data, as illustrated in FIGS. 8A to 8C demonstrates that
SNP treatment was effective at removing mixed species biofilms
established in a model potable water distribution system as well as
S. marcescens in a dose dependent manner. Consistent results were
obtained from viability assays and microscopy analysis (FIG. 8).
The relative proportions of different colony morphologies on the
plates were not affected by the SNP treatments, suggesting that
this treatment was not selective for specific species within the
mixed community. The most efficient treatment concentration was 500
nM SNP, which correlates with previous results observed with
Pseudomonas aeruginosa and other monospecies bacterial biofilms. In
this experiment, free chlorine treatment was used at 2 ppm and
complete removal of biofilms was observed on all coupons that were
exposed to conventional chlorine.
[0154] 5.2.2 Recycled Water Biofilms Spiked with Serratia
marcescens
[0155] Mixed species biofilms established from a model recycled
water distribution system and harboring S. marcescens were also
reduced in both total counts and S. marcescens counts upon exposure
to nanomolar concentrations of the NO donor SNP in a dose dependent
manner. Consistent results were obtained from viability assays and
microscopy analysis (FIG. 9). Biofilms exposed to 500 nM SNP also
exhibited increased sensitivity to free chlorine treatments and,
for example, 1 ppm free chlorine was up to 20-fold more efficient
at removing SNP treated biofilm compared to control biofilms as
determined by viability counts (FIG. 9).
Example 6
Low Levels of Nitric Oxide to Induce Dispersal of S. marcescens, V.
Cholerae, E. coli and B. licheniformis Biofilms
[0156] Bacterial biofilms were grown in petri dishes (90 mm
diameter) containing either glass (Superfrost, Menzel Glaser) or
polycarbonate microscope glass slides (76.times.26 mm,). To prevent
contamination, the slides were autoclaved (glass) or sterilized in
a 1% solution of bleach for 30 minutes and the petri dishes were
sterilised by 30 minutes exposure to ultraviolet light. Overnight
cultures of bacteria were diluted 1/1000 into 25 ml of fresh medium
and grown for 24 hours, at 30.degree. C. or 37.degree. C. with 50
rpm shaking, allowing biofilm formation on the slides. After 24 h,
the medium was replaced with fresh medium containing varying
concentrations of SNP, SNAP, or GSNO (in addition to controls
without NO generators) and the cells were incubated for a further
24 hours at the appropriate temperature with agitation at 50 rpm.
The slides were then rinsed in sterile PBS to remove unattached or
loosely attached cells.
[0157] The ability of SNP to increase the sensitivity of V.
cholerae biofilms to antimicrobial treatments was also tested as
described above, with the exception that, after the initial 24
hours of biofilm development, the NO donor was added in combination
with the anti-microbial treatment. Controls included a no treatment
control, and the antimicrobial alone and the cells were incubated
for a further 24 hours at the appropriate temperature with
agitation at 50 rpm. The slides were then rinsed in sterile PBS to
remove unattached or loosely attached cells. All treatments were
performed in triplicate.
[0158] Assessment of biofilm formation was performed by staining
the cells with the BacLight Live-Dead Staining reagents (Molecular
Probes Inc, USA) and subsequent confocal microscopy. Up to 15
randomly selected fields of view per slide were imaged in the x-y
plane for subsequent image analysis. Image analysis was performed
using the analysis package, ImageJ (http://rsb.info.nih.gov/ij) to
determine total surface coverage. Results are presented as
percentage cover of the total surface available per field of
view.
[0159] FIG. 10 shows a concentration dependent dispersal of S.
marcescens biofilms by SNP at concentrations between 0 and 500 nM,
with more than 60% reduction in biofilm coverage at a concentration
of 25 nM SNP. FIG. 11 shows that SNAP (100 nM) is also effective
for dispersal of S. marcescens biofilms.
[0160] FIGS. 12 and 13 show similar results for the effects of SNP
and SNAP on dispersal of Vibrio cholerae biofilms.
[0161] FIG. 14 shows that SNP enhances the antimicrobial activity
of tetracycline (6 .mu.g/mL) on biofilms of V. cholerae. The
concentration of tetracycline used was below the MIC for this
organism.
[0162] FIG. 15 shows that 1 .mu.M GSNO has a significant effect on
stability of V. cholerae biofilms, and FIG. 16 shows that 500 nM
SNP has a significant effect on stability of E. coli biofilms.
[0163] FIG. 17 shows a strong effect of SNP on stability of
Bacillus licheniformis biofilms, with 100 nM SNP providing a 90%
reduction in surface coverage by the biofilms.
Example 7
Low Levels of Nitric Oxide Induce Dispersal of C. albicans
Biofilms
[0164] Cells were grown in 24 well polystyrene microtitre plates
(Sarstedt) in Yeast peptone dextrose medium (YPD) at 30.degree. C.
with shaking at 100 rpm. Briefly, an overnight culture of C.
albicans was diluted 1:100 into fresh medium and 1 ml inoculated
into the wells. Biofilm was allowed to form for 24 hours, after
which time the medium was replaced with fresh medium and SNP was
added at concentrations of 0 nM, 25 nM, 100 nM, 500 nM, 1 .mu.M and
5 .mu.M. The cells were incubated a further 24 hours, at which time
the wells were rinsed with PBS to remove loosely and unattached
cells and stained with 1% crystal violet. The wells were washed
thoroughly with PBS and the amount of crystal violet absorbed into
the biofilm was measured using a Wallac-Victor.sup.2 plate reader
(Perkin-Elmer) at 540 nm.
[0165] The results (FIG. 18) are presented as the percentage of the
untreated control and show that SNP destabilises C. albicans
biofilms at SNP concentrations below 1 .mu.M, with more than 60%
reduction in C. albicans biofilm in the 25 nM SNP treatment.
Example 8
Low Levels of Nitric Oxide Inhibit Formation of S. epidermidis
Biofilms
[0166] The methods and materials used to test the effect of NO on
biofilm formation and develop of Staphylococcus epidermidis were
similar to those described above for S. marcescens, V. cholerae, E.
coli and B. licheniformis, using glass slides in petri dishes for
cultivation of the biofilm. However, the NO donor, SNP, was added
continuously to the cells, rather than after the initial 24 hours
period of biofilm development. Results demonstrate that SNP
addition can prevent the formation of a biofilm by S. epidermidis
(FIG. 19) in a concentration-dependent manner.
Example 9
Low Levels of Nitric Oxide Induce Dispersal of F. nucleatum
Biofilms
[0167] To determine the potential effect of NO on anaerobic, oral
bacteria, Fusobacterium nucleatum was selected as a model and key
organism for biofilms of oral consortia. Briefly, an overnight
culture was used to inoculate fresh medium (1:100). The cells were
grown to an optical density (600 nm) of 0.1, at which time SNP was
added to the cells to a concentration of 0 nM, 100 nM, 500 nM, 1
.mu.M and 10 .mu.M. A glass slide was also added to the tube of
cells, and the bacteria were allowed to attach for 4 hours. At the
end of the incubation period, the slides were removed, washed twice
by dipping in sterile PBS to remove loosely associated cells, and
stained with crystal violet, The attached cells were enumerated
microscopically by digital image capture and subsequent image
analysis. Results are presented as the percentage of cells attached
in comparison to the control culture which was not exposed to SNP
(FIG. 20), and show that addition of the NO generator, SNP,
inhibits attachment of F. nucleatum to surfaces.
[0168] It will be appreciated that, although specific embodiments
of the invention have been described herein for the purpose of
illustration, various modifications may be made without deviating
from the spirit and scope of the invention as defined in the
following claims.
* * * * *
References