Note: Descriptions are shown in the official language in which they were submitted.
1
PROCESS FOR REMOVAL OF BIOFILM
[1] [Intentionally left blank].
Technical Field
[2] The present invention relates to a process for removing dry surface
biofilm
from a surface.
Background of Invention
[3] In general, biofilms are composed of microorganisms attached to
surfaces
and encased in a hydrated polymeric matrix of their own synthesis. The matrix
is
composed of polysaccharides, proteins, and nucleic acids which are
collectively termed
"extracellular polymeric substances" (EPS). The EPS matrix enables cells in a
biofilm to
stick together and is a key element in the development of complex, three-
dimensional,
attached communities. Water channels are dispersed throughout biofilms,
allowing the
exchange of nutrients, metabolites, and waste products.
[4] Biofilms form virtually anywhere there is water. Sites include
inorganic natural
and manmade materials above and below ground, on minerals and metals,
including
medical implant materials, and on organic surfaces such as plant and body
tissues.
Biofilm growth surfaces may act as an energy source, a source of organic
carbon, or
simply a support material. One common feature of biofilm environments is that
they are
periodically or continuously suffused with water.
[5] One common example of a biofilm dental plaque, a slimy build-up of
bacteria
that forms on the surfaces of teeth. Similarly, the slimy layers often found
on rocks in
rivers and streams are also formed from biofilm.
[6] Biofilms cause a significant amount of all human microbial infections.
Nosocomial (hospital acquired) infections are the fourth leading cause of
death in the
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2
U.S. with 2 million cases annually (or approximately 10% of American hospital
patients) leading to more than $5 billion in added medical cost per annum.
About 60-
70% of nosocomial infections are associated with some type of implanted
medical
device. It is estimated that over 5 million medical devices or implants are
used per
annum in the U.S. alone. Microbial infections have been observed on most, if
not all,
such devices, including: prosthetic heart valves, orthopaedic implants,
intravascular
catheters, artificial hearts, left ventricular assist devices, cardiac
pacemakers,
vascular prostheses, cerebrospinal fluid shunts, urinary catheters, ocular
prostheses
and contact lenses, and intrauterine contraceptive devices.
[7] Until fairly recently, the general consensus was that biofilm needed a
moist
or wet environment in order to develop. Normally dry surfaces were thought not
to
form bacterial biofilm. However, a study by Vickery et al (Reference 1) showed
that
biofilm could be found on normally dry surfaces. These biofilms were found to
contain
multiple bacteria, including Pseudomonas spp., Staphylococcus aureus,
Enterococcus faecium, etc.
[8] In this study, Vickery destructively sampled items within a
decommissioned
hospital intensive care unit (ICU) after it was terminally disinfected by
initially cleaning
with neutral detergent, followed by disinfection with 500 ppm chlorine.
Following
disinfection, equipment and furnishings were aseptically removed from patient
and
common-use areas.
[9] Items removed were then destructively sampled using sterile gloves,
forceps, pliers, scissors, or scalpel blades, depending on the material being
sampled.
Gloves and instruments were changed between each sample. Samples were then
placed into sterile containers for transport to the laboratory. Small items,
such as a
.. sterile supply reagent box, were transported intact to the laboratory;
larger items,
such as the mattress and door, had sections removed into sterile containers.
Following transport to the laboratory, these large pieces were further
sectioned into
smaller pieces, using a sterile technique.
[10] Samples were the examined by Scanning Electron Microscopy (SEM).
Biofilm was found on 5 out of 6 samples examined. Four samples had principally
3
coccoid-shaped bacteria encased in large amounts of EPS and the sample from
the
curtain had 'strings' of dehydrated EPS evident.
[11] Bacteria grew on Horse Blood Agar plates from four of the six samples,
demonstrating the presence of culturable organisms. Samples taken from a
venetian
blind cord and curtain, shown to be positive for biofilm by SEM, also grew
Methicillin
Resistant Staphylococcus aureus (MRSA).
[12] Re-examination of these samples after 12 months of storage under dry
conditions was shown to still have viable bacteria present (Reference 2), with
many of
the samples still demonstrating the presence of drug resistant organisms such
as
MRSA, Vancomycin Resistant Enterococcus (VRE), Extended Spectrum Beta
Lactamase (ESBL) producing organisms etc.
[13] The fact that the presence of these dry surface biofilms is present in
the
hospital environment strongly suggests that they may serve as a reservoir for
these
resistant organisms, thus play a role in the prevalence of nosocomial
infections was
further provided in a study by Whiteley et a/ (Reference 3), in which the
location of
potential dry surface biofilm was determined using ATP swabbing, and the
presence of
resistant organisms confirmed by microbial culturing. A further study, as yet
unpublished, was able to demonstrate that the organisms found in dry surface
biofilm in
the ICU environment were very closely related to isolates taken from patient
found to be
colonised with Multiply Resistant Organisms (MRO's).
[14] It was hypothesised (see Reference 1) that dry surface biofilm can
develop
where surface condensation occurs, producing a thin film of water, or that the
relative
humidity in the ICU is high enough to allow biofilms to develop on ICU
surfaces. Once
formed, the EPS would protect the bacteria from desiccation and make them
harder to
remove.
[15] It was further hypothesised that Multiply Resistant Organisms persist
in the
environment, in the face of enhanced cleaning, as biofilms. Although
detergents are
good at removing patient soil and planktonic bacteria, they are less effective
at
removing biofilm, rendering current cleaning protocols less efficient.
Date Recue/Date Received 2021-09-08
4
[16] Another potential route to the development of dry surface biofilm on
environmental high touch surfaces could be the deposition of proteinaceous
solutions
arising from various bodily fluids (sweat, saliva, blood) onto the
environmental surface,
thus allowing early colonisation by opportunistic biofilm forming micro-
organisms.
Repeated contact of the high touch surfaces may provide intermittent nutrients
to the
dry surface biofilm.
[17] Following on from this discovery of Dry Surface Biofilm (DSBF), a
laboratory
model was developed by Almatroudi et al (Reference 4).
[18] Normal wet surface biofilm is typically grown in a CDC Biofilm
reactor:
following a standard method as described in ASTM E2562 (see Reference 5).
Almatroudi modified the methodology used in ASTM E2562 to generate dry surface
biofilm by incorporating prolonged periods of dehydration in between exposure
of the
sample coupons to growth media. In this way, the Almatroudi methodology
attempts to
replicate the conditions under which dry surface biofilm is thought to grow
(i.e.,
exposure of the surface to occasional aqueous nutrients (cleaning chemicals,
biological
fluids etc) followed by extensive periods of desiccation).
[19] Examination of the model dry surface biofilms were compared to those
dry
surface biofilms recovered from dry environmental surfaces and were shown to
have a
similar morphology and composition.
[20] Both model and environmental dry surface biofilms were also found to
differ
from conventional wet surface biofilms.
[21] Firstly, whilst the EPS of conventional biofilm (i.e., those found in
normal, wet
environments) tend to be predominantly formed from polysaccharides, the EPS of
dry
surface biofilm (DSBF) is notably richer in protein.
[22] Secondly, whilst it is well known that the conventional wet surface
biofilm
forms a very protective environment for the bacteria embedded within the
biofilm, which
serves to protect the embedded bacteria from biocides such as disinfectants,
antimicrobial drugs etc, dry surface biofilm appears be significantly more
protective.
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[23] For example, Almatroudi eta/have also demonstrated that organisms
within dry surface films were remarkably resistant to treatment with chlorine,
with a
Staphylococcus aureus dry surface biofilm still showing survivors after
exposure to
sodium hypochlorite solution containing 20,000ppm available chlorine
(Reference 6).
5 [24] Similarly, it has also been demonstrated that subjecting dry-
surface biofilm
to dry heat (up to 121 C for 20 minutes) had minimal effect on the bacteria
embedded
within the dry surface biofilm, reducing bacterial numbers by only 2 logio
whilst
planktonic cultures and hydrated biofilm counts were reduced over 8 logio and
7 logio,
respectively. It was further shown that it is possible to recover viable
organisms after
autoclaving at 121 C for up to 30 minutes (Reference 7).
[25] In more recent, as yet unpublished, studies into the proteomics of the
various forms of biofilm produced by Staphylococcus aureus, significant
differences in
the proteins upregulated when forming differing biofilms were observed
compared to
the planktonic form (see Table 1 and Figure 4). The differences in protein
makeup
between the various forms of biofilm are likely to account for the observes
and
reported differences in resistance to biocides such as chlorine, temperature
and
prolonged storage in the desiccated state.
Table 1: Proteomic study of various biofilm of Staphylococcus aureus
Number of Distinct proteins Exclusive or common Biofilm type
52 Exclusively 3 Day wet biofilm (3DWB))
33 Exclusively 12 Day wet biofilm (12DWB)
26 Exclusively 12 Day dry biofilm (12DDB)
15 Common 3DWB + 12DWB
7 Common 3DWB + 12DDB
38 Common 12DWB + 12DDB
47 Common 3DWB + 12D WB + 12DDB
[26] It is evident therefore that the dry surface biofilm described in
References
1, 2, 3, 4, 6 and 7 represent a hitherto unrecognised surface colonisation
mechanism
available to many bacteria, and that this dry surface biofilm provides its
embedded
bacteria with enhanced protection against desiccation, exposure to biocides
and even
exposure to extreme temperature compared to the widely recognised wet biofilm.
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The presence of dry surface biofilm within healthcare facilities also clearly
poses an
increased risk of nosocomial infections by serving as a reservoir for
pathogenic, drug-
resistant organisms.
[27] Given the increased resistance of organisms within a dry surface
biofilm,
there is a clear need for a means of removing the dry surface biofilm from a
contaminated surface, and also killing the embedded bacteria. It is clear that
the
standard methods employed within healthcare establishments currently are
ineffective
against dry surface biofilm, as evidenced by the recovery of viable MRO's
following
terminal cleaning.
[28] It has been unexpectedly found that a disinfectant product, based on a
powder formulation that is dissolved in water prior to use has proven to be
efficacious
in both destroying bacteria within a dry surface biofilm, and also in
substantially
removing the protein present in the dry surface biofilm
Summary of Invention
[29] Described herein is a process of removing dry surface biofilm from
both
environmental surfaces (floors, walls etc) as well as from non-critical
medical devices
such as bedframes, infusion pump stands, infusion pump keyboards etc).
[30] According to a broad form of the invention there is provided a
process for
removing dry surface biofilm from a surface, which process comprises:
(i) dissolving a powder-based composition into water wherein the
powder-based composition comprises:
a) a hydrogen peroxide source
b) an acetyl donor
c) an acidifying agent, and
d) a wetting agent
(ii) allowing the solution to generate a biocidally effective concentration
of peracetic acid;
(iii) contacting the dry surface biofilm contaminated surface with the
solution of peracetic acid for a period of time; and
(iv) removing the solution.
7
[31] Where the terms "comprise", "comprise" or "comprising" are used in
this
specification (including the claims) they are to be interpreted as specifying
the presence
of the stated features, integers, steps or components, but not precluding the
presence
of one or more other features, integers, steps or components, or group
thereof.
[32] The terminology "biocidally effective" is to be taken as meaning a
substance
that will effectively kill, inactivate or repel living or replicating
organisms, including
spores, bacteria, fungus, virus, yeasts and moulds. A solution of the
composition
described herein is particularly effective as a sporicide. A solution of the
composition
described herein is also effective against viral species, particularly blood
borne viruses
such as HIV, Hepatitis A, B and C. The invention will also be active against
other viral
species such as filoviruses (e.g., Ebola, Marburg) and arenavirus (Lassa),
even in the
presence of whole blood. The fact that peracetic acid is not deactivated by
catalase
makes the composition particularly useful against these latter haemorrhagic
fever
inducing species.
Brief Description of Drawings
[33] Figure 1 is a graph showing the variation in concentrations of
hydrogen
peroxide and peracetic acid with time following dissolution of the composition
described
herein in tap water.
[34] Figure 2 is a graph showing the variation in concentrations of
peracetic acid
with time following dissolution of differing weights of the composition
described herein in
tap water.
[35] Figure 3 is a graph showing the peracetic acid (PAA) concentration
generated for various samples of sachets of the composition described herein,
dissolved in tap water, at 10 minutes, 20 minutes and 30 minutes.
[36] Figure 4 shows a Venn diagram outlining the differences in numbers of
distinct upregulated proteins in various biofilms of Staphylococcus aureus.
[37] Figure 5 shows the results of a Crystal Violet assay for the removal
of wet
biofilm using differing cleaning products
Date Recue/Date Received 2021-09-08
8
[38] Figure 6 shows the log reduction obtained from a disinfectant
according to
Example 9, Chlorclean nil and sodium dicloroisocyanurate (SDIC) under both
clean and
dirty conditions.
[39] Figure 7 shows the protein removal from a dry surface for a
disinfectant
according to Example 9, 1000ppm chlorine (sodium hypochlorite) and 1000pm
chlorine
(SDIC).
[40] Figure 8 shows the bacterial reduction of a range of disinfectants
against
planktonic Staphylococcus aureus.
[41] Figure 9 shows the bacterial reduction of a range of disinfectants
against dry
surface biofilms formed by Staphylococcus aureus.
Detailed Description
[42] It has unexpectedly been discovered that the disinfecting composition
described in the applicant's earlier United States Application no. 15/035,633
('633) may
be used as a dry surface biofilm remover.
[43] US 15/035,633 describes a composition which, on dissolution in a
solvent,
generates a biocidally effective disinfectant solution comprising peracetic
acid and
hydrogen peroxide. The composition comprises a system to produce a visual
indication
of the formation of the peracetic acid. The indication is provided by a dye
that is rapidly
bleached in the presence of peracetic acid, whilst being substantially
unaffected by the
presence of hydrogen peroxide. An optional second dye may be incorporated,
wherein
the second dye is not substantially bleached by either peracetic acid or
hydrogen
peroxide.
[44] Preferably the composition of '633 is provided in a powder. Preferably
the
composition of '633 is dissolved in water.
[45] When the composition of '633 is presented in powdered form, it may
also
contain a flow modifier to prevent clumping of the powder prior to dispersion
and
dissolution into the solvent, and a wetting agent to assist in the rapid
dispersion and
dissolution of the acetyl source into solution, preferably at ambient
temperature.
Date Recue/Date Received 2021-09-08
9
[46] The composition of '633 may also be packaged into a soluble sachet
wherein
the entire sachet and contents is placed into a solvent, preferably water, to
generate the
disinfectant, thus mitigating occupational exposure to the potentially harmful
powder
precursor.
[47] In a preferred embodiment of '633, there is provided a composition
comprising a hydrogen peroxide source, an acetyl donor, an acidifying agent,
and a first
dye that is bleached in the presence of peracetic acid, but not hydrogen
peroxide. In
another embodiment, a second dye that is substantially bleach-stable may also
be
included in the composition of '633.
[48] In a particularly preferred embodiment of '633, the first dye is a dye
that is
bleached in the presence of a biocidel concentration of peracetic acid, and
the second
dye is a dye that is bleached after several hours in the presence of a
biocidal
concentration of peracetic acid. The presence of the first dye in the solution
acts as a
visual indication that the solution has not yet achieved the desired biocidal
concentration of peracetic acid. Once the colour due to the first dye is
discharged, the
colour due to the second dye is left to provide an aesthetically pleasing
colouration.
When the composition of '633 is in powder form, it is dissolved in a solvent,
preferably
water, to form the peracetic acid-containing solution.
[49] The composition of '633 may also optionally contain wetting agents,
sequestering and chelating agents, and other ingredients, such as bleach-
stable
fragrances, corrosion inhibitors, powder flow modifiers, rheology modifiers
etc.
[50] The composition of '633 is prepared by combining the ingredients
together. In
a preferred embodiment, the composition of '633 is in powder form.
[51] In an alternative embodiment, the composition of '633 may be presented
in
kit form, where the hydrogen peroxide source, part (a), is stored separately
to a mixture
of the acetyl source and peracetic acid bleachable dye, parts (b) and (c). In
use, the
hydrogen peroxide source is mixed with the acetyl source/peracetic acid
bleachable dye
mixture, in solution.
[52] In use, the composition of '633 is dissolved in a solvent and to
produce a
broad-spectrum disinfectant solution which is efficacious against spores,
bacteria
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PCT/IB2018/001437
fungus, virus, yeasts and moulds. The disinfectant solution is particularly
efficacious
against spore forming bacteria such as Clostridium difficile. The disinfectant
may be
used to disinfect surfaces, including hard surfaces, and instruments.
[53] It has unexpectedly been discovered that the disinfecting composition
5 described in '633 may be used as a dry surface biofilm remover.
[54] When a surface coated in a dry surface biofilm is contacted with a
solution
of peracetic acid generated by dissolving the compositions taught in '633 it
has been
observed that there is a significant reduction in viable bacteria, along with
a
substantial removal of the protein typically associated with the dry surface
biofilm.
10 [55] This
observation is all the more remarkable given that detergent solutions
demonstrated to remove normal, wet surface biofilm have very little effect in
removing
dry surface biofilm (see Example 10). In this screening test it was also
observed that
chlorine-based disinfectants were also effective in removing dry surface
biofilm under
clean conditions. However further testing showed that the presence of an
organic
proteinaceous soil rapidly deactivated the chlorine, and thus resulted in
little or no
bacterial kill (see Figure 6). It was also observed that the chlorine-based
disinfectants
gave a lower removal of protein from dry-surface biofilm coated surfaces
compared to
the '633 solution (see Figure 7). These observations are consistent with the
observation of dry surface biofilm being found on samples removed from a
decommissioned hospital Intensive Care Unit, even after terminal cleaning with
a
chlorine-based disinfectant (see reference 1).
[56] The disinfecting composition described in the '633 document is a
powder-
based formulation comprising a hydrogen peroxide donor, and acetyl donor,
along
with acidifying agents, wetting agents, along with optional ingredients such
as
additional sequestrants and perfumes.
[57] The compositions of '633 also contain a peracetic acid (PAA)
bleachable
dye to serve as an indicator as to when a biocidally active concentration of
peracetic
acid has been generated. For the avoidance of confusion, a biocidally active
concentration of peracetic acid is defined as a concentration of peracetic
acid above
1300ppm.
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[58] Whilst the teachings of '633 are directed towards peracetic acid
generating
compositions containing an indicator system comprising a peracetic acid
bleachable
dye, a person generally skilled in the art will recognise that the presence or
absence
of this indicator will not affect the biocidal performance of the peracetic
acid
generating compositions.
[59] The present invention is directed to a process for removing dry
surface
biofilm from a surface.
[60] According to the present invention, there is provided a process
for
removing dry surface biofilm from a surface, which process comprises:
(i) dissolving a powder-based composition into water wherein the powder-
based composition comprises:
a) a hydrogen peroxide source
b) an acetyl donor
c) an acidifying agent, and
d) a wetting agent
(ii) allowing the solution to generate a biocidally effective concentration
of
peracetic acid;
(iii) contacting the dry surface biofilm contaminated surface with the
solution
of peracetic acid for a period of time; and
(iv) removing the solution.
[61] In other preferred embodiments, the powder-based formulation may
be in
the form of a tablet. In this case, the composition may also contain
disintegrants. An
example of a tabletted formulation is given in example 16 of '633.
[62] Typically, the composition of '633, as used in the process of the
present
invention, contains the following ingredients:
[63] Hydrogen Peroxide Source
[64] Examples of a hydrogen peroxide source which may be used in the
composition of '633 and in the present invention include, but are not limited
to, sodium
perborate, sodium percarbonate, urea peroxide, povidone-hydrogen peroxide,
calcium peroxide, and combinations thereof.
12
[65] A dilute solution of hydrogen peroxide in water may also be used as a
hydrogen peroxide source, if a two-part product is intended. In this case, the
hydrogen
peroxide solution should preferably contain less than 8% hydrogen peroxide,
thus
negating classification as a Class 5.1 Dangerous Good. The dilute solution of
hydrogen
peroxide may also contain additional stabilising ingredients, such as 1-
hydroxyethylidene -1,1,-diphosphonic acid, (sold as DequestTM 2010), or other
strongly
chelating additives, such as ethylenediamine tetraacetic acid (EDTA). The
peroxide
solution may optionally contain pH buffering agents.
[66] Acetyl Donors
[67] Examples of acetyl donors which may be used in the composition of '633
and
in the present invention include, but are not limited to,
tetraacetylethylenediamine
(TAED), N-acetyl caprolactam, N-acetyl succinimide, N-acetyl phthalimide, N-
acetyl
maleimide, penta-acetyl glucose, octaacetyl sucrose, acetylsalicylic acid,
tetraacetyl
glycouril, and combinations thereof. Preferably the acetyl donor is a solid.
The acetyl
donor is understood as being an uncoated material unless otherwise indicated.
[68] A preferred acetyl donor is TAED, more particularly, a micronized
grade of
TAED, such as B675, obtainable from Warwick Chemicals (UK).
[69] Acidifying agents
[70] Examples of acidifying agents which may be used in the composition of
'633
and in the present invention include, but are not limited to, citric acid,
monosodium
citrate, disodium citrate, tartaric acid, monosodium tartrate, sulfamic acid,
sodium
hydrogen sulphate, monosodium phosphate, oxalic acid, benzoic acid,
benzenesulfonic
acid, toluenesulfonic acid and combinations thereof. Preferably the acidifying
agent is a
solid.
[71] Peracetic acid bleachable dyes
[72] The 'first dye' is a peracetic acid bleachable dye. Examples of
peracetic acid
bleachable dyes which may be used in the composition of '633 and in the
present
invention include Amaranth (C.I. 16185), Ponceau 4R (C.I. 16255), FD&C Yellow
6 (C.I.
15985), any other 1-arylazo-2-hydroxynaphthyl dye, and combinations thereof.
Date Recue/Date Received 2021-09-08
13
[73] The peracetic acid bleachable dye is preferably relatively rapidly
bleached in
the presence of peracetic acid, but not hydrogen peroxide. By "relatively
rapidly" is
meant that the colour of the dye is bleached within about 10 minutes. When the
colour
generated by the peracetic acid bleachable dye in solution is substantially
discharged,
the peracetic acid has reached a biocidally effective concentration in the
solution. By
"substantially discharged" is meant that the colour in the solution, generated
by the
peracetic acid bleachable dye, is entirely, or almost entirely, discharged.
[74] In a preferred embodiment of the composition of '633, as used in the
present
invention, the first dye is Amaranth Red (C.I. 16185) and the second dye is
C.I. Acid
Blue 182. Surprisingly, it has been found that in this embodiment, Amaranth
Red is
bleached rapidly by only peracetic acid, whilst being relatively resistant to
bleaching by
hydrogen peroxide. This is a particularly unexpected finding, as Amaranth Red
is used
as an indicator in a commercially available powder-based detergent called
Virkon TM a
product produced and marketed by Antec Ltd. In the case of Virkon TM , as long
as the
red colouration due to Amaranth is present, the Virkon TM solution is still
actively biocidal.
According to the Virkon TM product brochure, "VIRKON TM 1% solutions are
stable for 7
days but should be discarded when the pink colour fades".
[75] Virkon TM is comprised of a mixture of potassium monoperoxysulfate,
sodium
chloride, sulfamic acid, plus other ingredients such as surfactants, perfumes,
as well as
Amaranth. According to a background document produced by Antec, on dissolution
in
water, the Virkon TM powder mix undergoes the Haber-Willstatter Reaction,
producing a
mix of biocidal species including the potassium monoperoxysulfate, chlorine, N-
chlorosulfamic acid, hypochlorous acid. The document goes on to state that
Virkon TM
contains "a pink dye (amaranth colour, EEC No. 123). In addition to being
aesthetically
pleasing, this serves a very practical purpose - it indicates whether the
VIRKONTM
solution is active. In its oxidised form, it is pink but when the solution
starts to lose its
activity it reverts to its colourless reduced form. VIRKONTM solutions must
always be
replaced if the colour starts to fade". In other words, the pink-red
colouration due to
Amaranth is present whilst the active oxidatively biocidal species are also
present, with
the colour only fading as the oxidative biocides become depleted.
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14
[76] Conversely, in '633, colour depletion of the disinfectant solution
indicates
that an effective biocidal concentration of peracetic acid has been achieved.
[77] Substantially bleach-stable dyes
[78] The second dye which may optionally be included in the composition of
'633, as used in the present invention, is a substantially bleach-stable dye.
It is
recognised that peracetic acid will be capable of bleaching most dyes, and
therefore
reference to a "substantially bleach-stable" dye is to be taken as meaning
that the dye
is capable of imparting colour to the peracetic acid/hydrogen peroxide
solution for at
least 2 hours, preferably about 4 to 6 hours, at room temperature.
[79] Examples of substantially bleach-stable dyes which may be used in the
composition of '633 and in the present invention include, but are not limited
to, Acid
Blue 182, Acid Blue 80, Direct Blue 86, Acid Green 25 (C.I. 61570) and
combinations
thereof.
[80] In a particularly preferred embodiment of the composition of '633, as
used
in the present invention, the first dye is Amaranth Red (C.I. 16185) and the
second
dye is C.I. Acid Blue 182. In this embodiment, the colour of the solution upon
dissolution of the composition is red, generated by the Amaranth. The red
colour
discharges at around 5-7 minutes, at which time the peracetic acid is at a
biocidally
effective concentration, leaving a blue colour, generated by the Acid Blue
182. The
blue colour is aesthetically pleasing, and has the added benefit of making the
solution
more visible when disinfecting a surface or object.
[81] Wetting agent
[82] When the composition of '633, as used in the present invention, is in
a
powder formulation, a wetting agent may be included in the composition to
facilitate
dispersion of the acetyl source into solution on initial dilution, thus
assisting in its
dissolution. The wetting agent is preferably comprised of a solid surfactant
capable of
lowering the surface tension of the solvent, preferably water, thus allowing
the acetyl
source to wet and disperse. Preferably, the acetyl source is TAED and, in the
absence of a wetting agent, a highly micronized grade of TAED such as B675
will
tend to float on the surface of the solvent, and thus be slow to dissolve,
resulting in
slow production of peracetic acid. Examples of suitable wetting agents which
may be
15
used in the composition of the invention include, but are not limited to,
sodium
dodececyl sulphate, sodium alkylbenzenesulphonate, PluronicTM PE6800, Hyamine
TM
1620 etc, and combinations thereof.
[83] pH buffering agents
[84] Optionally, a pH buffer may be included in the composition of '633, as
used in
the present invention, to reduce the variation of pH with time. Since the
formation of
peracetic acid from the acetyl source, preferably TAED, requires the pH to be
at, or
above, the pKa of peracetic acid (8.2), the pH of the solution should be
buffered
between 8.00 and 9.00, preferably between 8.00 and 8.40. Suitable pH buffers
which
may be included in the composition of the invention include, but are not
limited to,
phosphate, borate, bicarbonate, TAPS (3-
{[tris(hydroxymethyl)methyl]aminolpropanesulfonic acid), Bicine (N,N-bis(2-
hydroxyethyl)glycine), Tris (tris(hydroxymethyl)methylamine), Tricine (N-
tris(hydroxymethyl)methylglycine) and combinations thereof.
[85] Sequestering agents
[86] Optionally, the composition of '633, as used in the present invention,
may
include ingredients capable of complexing metal ions such as calcium and
magnesium,
thus negating any adverse effect from the use of hard water, as well as metal
ions such
as iron, manganese, copper etc which are capable of catalysing the
decomposition of
peroxides, and which also may be present in tap water. Examples of chelating
and
sequestering agents which may be used in the composition of the invention
include, but
are not limited to, sodium citrate, citric acid, phosphoric acid, sodium
tripolyphosphate,
EDTA, NTA, etc and combinations thereof.
[87] Flow modifiers
[88] A flow modifier may be added to improve the flow characteristics of
the
composition of '633, as used in the present invention, when in a powder
formulation.
This is particularly useful if the powder is intended for supply in a unidose
package (eg
an individual sachet or water soluble pouch), as good powder flow will allow
accurate
dosing of the blended powder into the individual packs. Examples of powder
flow
modifiers which may be used in the composition of '633 and in the present
invention
include, but are not limited to, fumed silica, precipitated silica, micronized
Date Recue/Date Received 2022-01-11
16
polyethylene glycol 6000, micronized lactose, talc, magnesium stearate etc,
and
combinations thereof.
[89] In a preferred example, the flow modifier is a hydrophilic fumed
silica, for
example AerosilTM 200 (Evonik Industries).
[90] It may also be possible to achieve good flow improvements using a
precipitated silica such as TixosilTm 38, although the precipitated silica
grades are less
preferred as they produce a strong haze in the final disinfectant solution, by
virtue of the
larger particle size of the precipitated form over the fumed form.
[91] Perfumes
[92] Optionally, the composition of '633, as used in the present invention,
may
also contain perfumes to mask the odour of peracetic acid. The perfume used
should
preferably be stable to hydrogen peroxide and peracetic acid.
[93] In a preferred embodiment of the composition of '633 and as used in
the
process of the present invention, the acetyl donor is TAED, the hydrogen
peroxide
source is sodium percarbonate, the first dye is Amaranth Red, and the
composition is in
a powder formulation, which is dissolved in water. On initial mixing of the
powder
formulation with tap water, at ambient temperatures, a deep red cloudy
solution is
formed by the rapid dissolution of the Amaranth Red dye and the suspension of
undissolved TAED. Over the course of approximately 5-10 minutes, the TAED
dissolves
into the water, and the red colouration is discharged as peracetic acid is
generated by
the reaction of the TAED with hydrogen peroxide produced by dissolution of the
sodium
percarbonate_ After about 7-10 minutes, the solution will be clear, and all of
the red
colouration discharged.
[94] In another preferred embodiment, a second dye that is substantially
bleach-
stable may also be included in the composition of '633, as used in the process
of the
present invention. Preferably the substantially bleach-stable dye bleaches
over the
course of 4-6 hours, along with the Amaranth. A preferred second dye, which is
slowly
bleached, is C.I. Acid Blue 182.
EXAMPLES
Date Recue/Date Received 2021-09-08
17
Example 1
[95] Dye premix: A mixture of 78.00g of TAED B675 (Warwick Chemicals),
17.00g
Amaranth dye and 5.00g of C.I. Acid Blue 182 dye were mixed and ground
together
using a pestle and mortar to give a homogenous brownish powder. Once mixed,
the dye
premix blend was stored in a well-sealed container prior to use.
[96] 54.55g of TAED B675, 1.00g of the dye-TAED premix, 1.32g of powdered
sodium dodecyl sulphate and 0.60g AerosilTM 200 (a hydrophilic fumed silica
available
from Evonik) were mixed together, and passed through a 125 micron sieve to
remove
and break up any aggregated material. After sieving, mixing was continued to
produce
a homogenous powder.
[97] To the sieved material was added 0.49g tetrasodium EDTA, 28.00g of
anhydrous citric acid, 99.32g of sodium percarbonate, 15.50g of sodium
tripolyphosphate and 1.80g of anhydrous monosodium phosphate. The powders were
then mixed thoroughly to produce a homogenous, free-flowing powder. The full
composition of the powder blend is shown in Table 2, along with the function
of each
ingredient.
[98] It was found that it was only necessary to add 1% of the TAED weight
of the
AerosilTM 200 to the powder blend. This equates to 0.3% of the overall blend
weight. At
this level, the Aerosil TM will produce only a very slight haze in the final
disinfectant
solution.
Table 2
Ingredient % w/w Function
Sodium percarbonate 49.03 Hydrogen peroxide source
TAED B675 27.31 Acetyl donor
Citric acid 13.82 Acidifier
Sodium tripolyphosphate 7.65 Sequestrant and pH modifier
Monosodium phosphate 0.89 pH modifier
Sodium dodecyl sulfate 0.65 Surfactant and wetting agent
Aerosil TM 200 0.30 Flow modifier
Tetrasodium EDTA 0.24 Chelating agent
Date Recue/Date Received 2021-09-08
18
Amaranth 0.084 PAA bleachable Colourant
Acid Blue 182 0.025 PAA stable Colourant
[99] A solution of the disinfectant was prepared by dissolving 7.50g of the
powder
blend into 500m1 of artificial hard water containing 340ppm CaCO3 (prepared as
described in SOP Number: MB-22-00: Standard Operating Procedure for
Disinfectant
Sample Preparation, published by the US Environmental Protection Agency Office
of
Pesticide Programs, and hereafter referred to as AOAC Hard Water). The
solution was
stirred at room temperature. The red colour due to the Amaranth was observed
to be
discharged at around 5-7 minutes, leaving a blue solution.
[100] 10m1 aliquots taken at regular intervals after 10 minutes, and the pH
were
also recorded. The aliquots were titrated to determine hydrogen peroxide and
peracetic
acid concentration.
[101] As may be seen in Figure 1, the concentration of peracetic acid
increases
rapidly, reaching its maximum value at around 20 minutes. After this point, a
slow
decay of the peracetic acid concentration over several hours is seen.
[102] Interestingly, if the concentration of powder dissolved into the
water is
increased, whilst the maximum peracetic acid concentration increases as
expected, it
was also observed that its decomposition rate was also increased (see Figure
1). It was
also observed that the maximum concentration of peracetic acid from each
powder
concentration was reached at the 20-minute mark.
Example 2
[103] 4 disinfectant solutions in AOAC Hard Water were prepared using
differing
concentrations of the powder blend from Example 1, and stirred for 20 minutes.
Aliquots
were taken and titrated for hydrogen peroxide and peracetic acid
concentration, whilst
further aliquots were inoculated with suspensions of both vegetative and spore
forms of
Clostridium sporogenes (ATCC 3584), in the presence of 5% horse serum. The
organisms were exposed for 3, 5 and 10 minutes. Each sample was tested in
triplicate,
and each sample gave greater than a 6-log reduction in viable organisms at
each time
point.
Date Recue/Date Received 2021-09-08
19
[104] Table 3 shows the concentration of the solutions used, the
concentrations of
both hydrogen peroxide and peracetic acid, along with the log reductions
recorded.
Table 3
Vegetative cells
Concentrations (ppm) Contact Time
H202 PAA 3 minutes 5 minutes 10 minutes
Sample 1 (20g/L) 1382 2964 >6 log >6 log >6 log
Sample 2 1330 2550 >6 log >6 log >6 log
(16g/L)
Sample 3 980 1980 >6 log >6 log >6 log
(12g/L)
Sample 4 (8g/L) 569 1349 >6 log >6 log >6 log
Bacterial spores
Concentrations (ppm) Contact Time
H202 PAA 3 minutes 5 minutes 10 minutes
Sample 1 (20g/L) 1382 2964 >6 log >6 log >6 log
Sample 2 1330 2550 >6 log >6 log >6 log
(16g/L)
Sample 3 980 1980 >6 log >6 log >6 log
(12g/L)
Sample 4 (8g/L) 569 1349 >6 log >6 log >6 log
Example 3
[105] 7.50g of the powder blend from Example 1 was taken, and added to
500m1 of
tap water, and stirred at room temperature. The time the red colour was
discharged
was noted, and a 5m1 aliquot taken and titrated. A further 5 ml aliquot was
removed and
titrated after 20 minutes.
[106] As can be seen in Table 4, the colour due to Amaranth was being
removed
between 7 and 8 minutes, with the peracetic acid content at this time being
between
0.14 and 0.16%.
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Table 4
Dye bleach
Time for Amaranth dye 20 min
Sample time
bleaching
HP PAA HP FAA
1 8 min 0.13% 0.14%
0.11% 0.21%
2 7min 50 sec 0.141 0.155 0.124
0.22%
3 7 min 0.13% 0.16%
0.11% 0.23%
[107] As can be seen in Table 3, solutions containing at least 1.35%
(1349ppm)
peracetic acid exhibit sporicidal activity, thus it may be safely assumed that
once the
5 red colouration due to Amaranth has been discharged, the peracetic acid
content will
be above this sporicidally active concentration.
Example 4
[108] Differing weights of the powder blend from Example 1 were taken, and
added to 500m1 of tap water, and stirred at room temperature. The time the red
10 colour was discharged for each solution is shown in Table 5.
Table 5
Weight of powder Weight of AOAC Hard Time taken for discharge of
(g) water (g) red colour
(minutes)
6.00 500 7.5
7.02 500.02 7
8.02 500.01 6.5
9.00 500 6
10.03 499.99 5.25
Example 5
[109] A quantity of the powder blend from Example 1 was taken, and packaged
15 into individual sachets prepared from heat sealed PVA water soluble
film. The
sachets were prepared by heat sealing two sheets of 50 micron thick PVA film
(width
4.65cm, length 8cm), together to form an envelope, dispensing approximately
8.2g
powder into each envelope and then sealing the open side to give the finished
filled
sachet.
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21
[110] A single sachet was then taken and added to a stirred quantity of tap
water
(500m1). The sachet was observed to wrinkle in the water, and then to burst
open,
releasing the contained powder into the water to give a deep red solution.
After
approximately 8 minutes, the red colour was discharged, leaving a pale blue
solution
with a faint odour of peracetic acid. Aliquots of the resultant solution were
taken at 10
and 20 minutes and titrated for hydrogen peroxide and peracetic acid content.
[111] Assessment of an initial production run to produce sachets is shown
in
Table 6, and Table 7 shows the result of assessing peracetic acid generation
from
several sample sachets dissolved into 500m1 tap water.
Table 6
mean sachet weight 8.29
Standard deviation 0.514
RSD 6.2
Maximum weight 9.59
Minimum weight 7.48
sample size 70
Table 7
10 minutes 20 minutes
sachet H202 PAA H202 PAA
pH pH
wt (g) 0/0 ok 0/0 OA
1 8.3 8.39 0.146
0.145 8.26 0.121 0.223
2 8.48 8.12 0.138 0.171 8.07 0.127 0.215
3 8.74 8.54 0.163 0.175 8.23 0.127 0.229
4 7.78 8.23 0.125 0.175 8.16 0.107 0.208
5 8.16 8.2 0.14 0.109 8.1 0.124 0.2
6 9.1 8.33 0.161
0.214 8.23 0.148 0.198
7 7.67 8.21 0.133 0.192 8.12 0.116 0.16
8 8.11 8.12 0.132 0.078 7.99 0.118 0.205
mean 8.29 8.27 0.14 0.16 8.15 0.12 0.20
Example 6
[112] A quantity of the powder blend according to Example 1 was taken, and
packaged into individual sachets prepared from heat sealed PET-paper-Aluminium-
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PP laminate. The sachets were prepared by heat sealing a sheet of laminate 6cm
wide to form a cylindrical tube, and then sealing across the tube to form a
stick, which
was then dosed with the powder blend via an auger doser. The open end of the
filled
tube was then sealed to give a stick pack.
[113] The mean gross weight of each stick pack was found to be 8.88g, with
a
standard deviation of 0.27 (see Table 8). The packaging material was found to
weigh
0.88g, thus giving a mean net weight for the powder of 8.00g.
Table 8
mean sachet weight 8.88
Standard deviation 0.27
c'/0 R S D 3.06
Maximum weight 9.66
Minimum weight 8.13
sample size 500
[114] To demonstrate homogeneity of blending, sample sachets were taken
from
various parts of a production run and added to 500m1 of tap water. The
hydrogen
peroxide and the peracetic acid content at 10, 20 and 30 minutes for each
solution
were then determined.
[115] As can be seen in Figure 3, the hydrogen peroxide and peracetic
acid
.. content at 10 minutes was highly variable, and was found to be dependent on
stirring
speed etc. In some cases, the solutions were observed to still be red at the
10 minute
mark (indicated by the letter R in Figure 3), and these solutions were all
associated
with a low peracetic acid content. It should be noted that by 20 minutes, the
variation
in concentrations of hydrogen peroxide and peracetic acid were significantly
reduced.
[116] This example demonstrates the utility of the dye system as an
indicator for
the presence of an effective biocidal concentration of peracetic acid.
[117] This was further illustrated by adding 7.50g of the powder blend
according
to Table 9 to 500m1 of AOAC Hard water and testing its biocidal activity
against
surface bound micro-organisms in an AOAC Hard Surface Carrier Test 991.47, 48
23
and 49, conducted in the presence of 5% horse serum against Pseudomonas
aeruginosa, Staphylococcus aureus and Salmonella choleraesuis. The test
methodology was modified to use a 5-minute contact time rather than the
prescribed 10-
minute contact time.
Table 9
Test organism No.
carriers No. Carriers No. Carriers Result
tested Negative Positive
Pseudomonas aeruginosa
60 57 3 PASS
ATCC 15442
Staphylococcus aureus
60 58 2 PASS
ATCC 6538
Salmonella choleraesuis
60 60 0 PASS
ATCC 10708
[118] The powder formulation can also be modified for the production of
tablets
capable of generating peracetic acid on dissolution into water. Preferably, a
means to
facilitate the disintegration of the tablet is incorporated into the tablet
formulation. This
also assists the slower dissolution of the tablet due to the compression
required to
generate the tablet.
[119] Poly-NVP based disintegrants such as DisintexTM 200 (ISP Technologies
Inc)
were found to be impractical for use, as the cross-linked polymer adsorbed the
dyes
strongly, and thus gave highly coloured particulate material in the final
solution. A
preferred means of disintegrating the tablet is to include additional sodium
carbonate
into the formulation, along with additional acidifying agent. In a more
preferred
embodiment, sulfamic acid is used as the acidifying agent as this lacks a pKa
above 2.
If citric acid is used as an acidifying agent in the tablet formulation, then
gas formation,
hence tablet disintegration, is slowed down once the solution reached a pH of
around 6
due to the third pKa of citric acid.
Example 7
[120] A powder blend according to Table 10 was produced by mixing the
ingredients together to produce a homogenous mix. In order to achieve adequate
Date Recue/Date Received 2021-09-08
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24
tablet formulation, the mixture was not sieved, and care was taken not to
reduce the
particle size of the soda ash, sodium percarbonate and the sulfamic acid.
Table 10
TAED 13.54
sodium
percarbonate 37.15
sulfamic acid 30.82
Dense soda ash 18.06
Sodium dodecyl
sulfate 0.23
Tetrasodium EDTA 0.15
Amaranth 0.038
CI Acid Blue 182 0.011
[121] Once blended, the material was tableted using a single punch tablet
press,
fitted with a 20mm die set to give tablets with a mean weight of 3.72g. The
mean
thickness of the tablets was 9.1mm, with a thickness to weight ratio of 0.41.
[122] Two tablets, with a combined weight of 8.34g were then dissolved in
200m1
of tap water. After stirring for 25 minutes at room temperature, three 10m1
aliquots
were removed and titrated. The mean concentrations for hydrogen peroxide and
peracetic acid were found to be 0.293% and 0.258% respectively.
[123] An additional tablet was taken and dissolved into AOAC Hard water,
and
then tested for antimicrobial activity against Clostridium difficile in the
presence of 5%
horse serum at 20 C, using the method of BS EN 1276 (1997). The resultant
observed log reductions are shown in Table 11.
Table 11
Test organism: Contact time (temperature = 20 C
Clostridium difficile
ATCC70992 1 5 10
Log reduction 2.77 >5.86 >5.86
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PCT/IB2018/001437
(initial inoculum level
4.8x106
Example 8: Production of model dry surface biofilm samples
[124] Dry
surface biofilm was produced in the surfaces of coupons following the
method described by Almatroudi et al. in Reference 4.
5 [125]
Staphylococcus aureus ATCC 25923 biofilm was grown on 24 removable,
sterile Pyrex coupons in an intensively cleaned, brushed and steam sterilised
(121 C
for 20 min) CDC biofilm reactor (BioSurface Technologies Corp, Bozeman, USA).
[126] Semi-dehydrated biofilm was grown over 12 days with cycles of batch
growth during which time 5% tryptone soya broth (TSB) was supplied alternating
with
10 prolonged dehydration phases at room temperature (22-25 C) as described
in Table
12, with the TSB being removed from the Biofilm Reactor at the end of each
batch
phase.
[127] The biofilm generator was located in an air-conditioned laboratory
and
filter-sterilised room air (average relative humidity 66%) was pumped across
the
15 media surface at an airflow rate of 3 l/min using an aquarium air pump.
[128] Biofilm development was initiated by inoculation of about 108 colony
forming units (CFU) of S. aureus at the beginning of the first batch phase.
During
batch phases, all biofilms were grown in 5% TSB at 35 C and subjected to
shear by
baffle rotation at 130 rpm/min producing turbulent flow.
20 Table 12
Stage Culture conditions Cumulative time
1 48 h batch phase in 5% TSB followed by 48 h 96 hr
dehydration
2 6 h batch phase in 5% TSB followed by 66 h 168 hr
dehydration
3 6 h batch phase in 5% TSB followed by 42 h 216 hr
dehydration
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26
4 6 h batch phase in 5% TSB followed by 66 h 288 hr
dehydration
[129] Following growth of the biofilm, the rods holding the biofilm coated
coupons
were removed from the generator, and placed in 1 litre of phosphate buffered
saline
(PBS) for 5 minutes. The three coupons on each rod were then removed, and
washed
an additional two times by placing them in to 50m1 PBS before being placed in
individual sterile Bijou containers. The number of CFU per coupon was
determined by
sonication of a randomly selected coupon in an ultrasonic bath (Soniclean,
JMR,
Australia) for 5 min and vigorous shaking for 2 min in 4 ml of media followed
by
sequential 10-fold dilution and plate count.
Example 9: Peracetic (PAA) based disinfectant
[130] A sachet containing 8.5g of a disinfectant powder composition similar
to
Example 1. The disinfectant powder comprised a blend of a hydrogen peroxide
source (sodium percarbonate) and an acetyl source (tetraacetylethylenediamine
(TAED)), along with acidifying agents (citric acid) and sequestrants
(monosodium
phosphate, sodium tripolyphosphate), along with a peracetic acid bleachable
dye
(amaranth). The formulation used is given in Table 13.
Table 13
Ingredient % w/w Function
Sodium percarbonate 49.18 Hydrogen peroxide donor
TAED B675 27.39 Acetyl donor
Citric acid 13.86 Acidifier
Sodium tripolyphosphate 7.67 Sequestrant
Sodium phosphate 0.89 pH modifier
Sodium dodecyl sulfate 0.65 Wetting agent
Tetrasodium EDTA 0.24 Chelating agent
Amaranth 0.08 PAA bleachable colourant
Acid Blue 182 0.03 PAA stable colourant
27
[131] The sachet was added to 500m1 water and stirred at room temperature
for
10-15 minutes, after which time the colouration provided by the peracetic acid
bleachable dye was discharged. At this point the solution will contain between
1500
and 2000ppm peracetic acid, along with about 1000-1300ppm hydrogen peroxide.
The
resultant solution was found to be active against a range of bacteria,
viruses, spores
and fungi for approximately 8 hours after dissolution.
Example 10: initial screening study using TOC to assess removal of dry surface
biofilm.
[132] In an initial screening study, a range of cleaning products were
assessed for
their dry surface biofilm removing efficacy by assessment of Total Organic
Carbon
(TOC). The products assessed, and their in-use concentrations are shown in
Table 14.
Table 14
Detergent Supplier Dilution
Fabrisan TM Whiteley Corporation Used undiluted
Matrix TM Whiteley Corporation 1:25
Zip Strip Whiteley Corporation 1:6
Phensol Whiteley Corporation 1:50
Example 9 Whiteley Corporation 17g per litre
Sodium hypochlorite Fronine Pty Ltd, 1000ppm available chlorine
1M Sodium hydroxide solution Chem Supply Ltd Used undiluted
Negative control (water) Used undiluted
[133] Fabrisan TM is marketed as a carpet spotter. Its ingredients include
sodium
citrate, sodium dodecyl sulfate, and Tea Tree Oil. The formulation is
according to
Example 3 of US patent no. 5610189
[134] Matrix TM is marketed as a wet surface biofilm remover. The
formulation is
according to Australian patent no. AU2001275599B2, and its efficacy against
normal
(wet) biofilm has been described by Vickery et al (Reference 8 and Reference
9), Ren
et al (Reference 10) and Fang et al, (Reference 11). The Ren and the Fang
references
were performed using Intercept, which has an identical formulation to MatrixTM
and is
manufactured under license from Whiteley Corporation by Medivators Inc.
Date Recue/Date Received 2021-09-08
28
[135] Zip Strip is a floor stripper intended to remove polymeric sealants
form vinyl
floors. The formulation comprises a highly alkaline solution of surfactants,
butyl glycol,
and ethanolamine.
[136] Phensol is a phenolic disinfectant comprising a blend of o-
phenylphenol and
benzyl chlorophenol with the sodium salt of a (C10-16) Alkylbenzenesulfonic
acid.
[137] Each cleaning solution was diluted according to the label directions,
as
shown in Table 14.
[138] A 12-day dry surface biofilm was grown on Pyrex TM glass coupons as
described in Example 8. Three coupons, coated in dry surface biofilm were then
placed
into 25m1 of each test product solution. Three coupons were also placed in
25m1 of Milli-
QTM water to serve as a negative control. AIM solution of sodium hydroxide was
used
as a positive control.
[139] Each sample was prepared and tested in duplicate.
[140] Blank coupons, in which fresh, clean coupons were exposed to the test
products were also produced, in order to assess any adherence of organic
materials
(such as surfactants) to the coupons, were also analysed.
[141] After exposure to the test product solution for the required time,
the coupons
were rinsed twice in 25m1 MilliQTM water. The Total Organic Carbon on each
coupon
was then measured using a Shimadzu-5000A TOC analyser. The TOC resulting from
any residual biofilm left after cleaning was calculated by subtracting the TOC
found on
the blank coupons from the TOC resulting from the residual carbon left on the
biofilm
coated coupons after cleaning.
[142] The results are given in Table 15. The percentage TOC remaining due
to the
biofilm shown in parentheses were calculated relative to the negative control
(Milli-QTm
water).
Date Recue/Date Received 2021-09-08
29
Table 15
TOC (pg)
Blank Coupons with TOC due to %
Reduction
coupons Biofilm biofilm
Fabrisan TM 1.13 4.77 3.64 51
Matrix TM 0.82 6.87 6.05 18
Zip Strip 1.16 5.33 4.17 43
Phensol 0.76 3.45 2.69 64
Example 9 0 0.47 0.47 94
Chlorine 100ppm 0.34 1.87 1.53 79
1MNaOH 0.17 0.16 -0.01 100
Negative control 0.06 7.43 7.37 0
[143] From this screening study, it can be clearly seen that products
demonstrated
to be efficacious in the removal of normal, wet surface biofilm (i.e.,
MatrixTM) do not
show the same degree of efficacy against dry surface biofilm. Apart from the
1M sodium
hydroxide solution, the two most efficacious cleaning solutions were Example 9
and
Chlorine.
Example 11
[144] The efficacy of removal of wet biofilm was assessed for both Example
9 and
Matrix TM a product demonstrated to remove wet surface biofilm.
[145] A wet Staphylococcus aureus biofilm was grown on plastic tiles
supported on
modified rods in a CDC Biofilm Reactor over 48 hours, following the
methodology of
Goeres et al (Reference 12).
[146] The plastic tiles were then placed into Falcon tubes containing
Matrix TM (at a
1:25 dilution in water), Example 9 (17g/L in water) and Milli-QTM water. The
tiles were
left immersed in the cleaning solutions for 10 minutes. After 10 minutes the
tiles were
removed, washed twice with Milli-QTM water, and then placed into 40 ml of a
0.3%
solution of Crystal Violet, (a stain for biofilm). The tiles were then stood
for 90 minutes in
the Crystal Violet solution. After 90 minutes, the tiles were removed, washed
for 1
minute three times in MilIiQTM water. The washed tiles were then scraped, and
eluted
Date Recue/Date Received 2021-09-08
30
with 5m1 of 95% ethanol into a 28m1 vial, which was then closed and stood
overnight to
elute the adsorbed Crystal Violet. The absorbances of the solutions were then
read via
a spectrophotometer.
Table 16
Cleaning product Absorbance
Example 9 0.128
Matrix TM 0.120
Milli-QTM water 0.191
[147] As can be seen in Table 16, Matrix TM removed most biofilm from the
tile as
shown by the lower absorbance due to Crystal Violet.
Example 12
[148] The efficacy of protein removal of Example 9 and Matrix TM was
demonstrated
as follows:
[149] 12 day biofilm was grown on PET coupons following the methodology of
Example 8. The rods containing the biofilm coated coupons were then removed
and any
loosely bound biofilm washed off with Milli-QTM water as described in Example
8.
[150] One rod holding three dry surface biofilm coated coupons was then
placed
into 30m1 of a solution of Example 9 (17g/litre) for 10 minutes. A second rod
was placed
into 30m1 of a solution of MatrixTM (1:50 dilution), and a third rod placed
into 30m1 Milli-
QTM water to serve as a positive control. An additional rod, holding 3
uncoated coupons
was sterilized and used as a negative control.
[151] After 10 minutes, each rod was placed into 30m1 of 1M sodium
hydroxide
solution to elute off all remaining protein. Aliquots from each solution were
then taken
and tested for protein using a Bicinchroninic Acid (BCA) assay, using a micro
BCATM
test kit (Sigma Aldrich).
[152] In order to perform the BCA assay, a series of standard solutions of
bovine
serum albumin were prepared to produce a standard curve. 1m1 of each of the
standard
BCA solutions, along with 1 ml aliquots taken from the cleaning solutions were
all then
treated with lml of a working BCA solution prepared by mixing 50m1
Bicinchononic acid
Date Recue/Date Received 2021-09-08
31
(Sigma Aldrich cat. B9643) into a beaker, and adding 1m1 of 4% copper (II)
sulfate
solution (Sigma Aldrich cat. No. C2284). The samples were then incubated for
60
minutes at 60 C, and the absorbance at 562nm read using a spectrophotometer
(see
Table 17).
Table 17
Sample Absorbance Derived concentration Percentage reduction of
Concn. BSA (PPm) protein
(PPm)
0 0
0.5 0.027
1 0.042
2.5 0.085
0.144
0.32
0.682
40 1.209
Test samples
Example 9 0.249 0.249 66.4
Matrix TM 0.362 0.362 50.5
Water 0.721 0.721 0.0
[153] As can be seen, Example 9 gives a significantly higher reduction of
protein
than MatrixTM when tested against 12 day dry surface biofilm.
Example 13
[154] The efficacy of protein removal from coupons coated in 12 day dry
surface
biofilm using Example 9, 1000ppm sodium hypochlorite solution and Chlorclean
TM , (a
sodium diisocyanurate (SDIC) tablet formulated with adipic acid and a sodium
toluenesulfonate and marketed as a 2-in-1 Hospital Grade Disinfectant with
detergent
action by Helix Solutions (Canning Vale South, Western Australia) were
assessed as
described in Example 11. Both chlorine solutions were shown to give 1000ppm,
available chlorine. In this test a 10-minute contact time was used. Percentage
reductions were calculated from the positive control (Milli-QTm water).
Date Recue/Date Received 2021-09-08
32
[155] As can be seen in Table 18, Example 9 gives the highest protein
reduction.
Chlorclean TM , a formulated SDIC tablet marketed as a 2-in-1
cleaning/disinfecting
product was also observed to be more efficacious than sodium hypochlorite
solution.
Table 18
Detergents tested Percentage protein
reduction
1000ppm Chlorine (sodium hypochlorite) 11.50
1000ppm Chlorine (ChlorcleanTM tablet) 39.26
Example 9 (17g/L) 63.65
Example 14
[156] In order to determine whether the presence of detergent moieties
within the
cleaning products were responsible for the difference in performance between
sodium
hypochlorite and the proprietary Chlorclean TM tablet, the methodology of
example 13
was repeated, only with the sodium hypochlorite solution being replaced with a
solution
of sodium diisocyanurate, giving 1000ppm available chlorine.
Table 19
Detergents tested Percentage reduction
1000ppm Chlorine (SDIC) 17.65
1000ppm Chlorine (ChlorcleanTM tablet) 13.12
Example 9 (17g/L) 64.69
[157] Of note here is the marked reduction in efficacy of Chlorclean TM
with the
shorter contact time. The protein reduction observed with the Example 9 was
observed
to be substantially the same despite the difference in contact times.
Example 15
[158] The bacterial reductions obtained from a 12-day dry surface biofilm
were
assessed under clean conditions using Example 9, ChlorcleanTM tablets and a
generic
SDIC tablet
[159] Each test product was dissolved in water.
Date Recue/Date Received 2021-09-08
33
[160] Coupons coated in 12-day dry surface biofilm were produced as per
Example
8. 2m1 of each test solution, followed by 2 ml of water were added to the
wells in a
tissue culture plate.
[161] After a 5 minute contact time, coupons were removed from the
disinfectant
solutions, rinsed twice using 30m1 phosphate buffered saline for 5 seconds,
and then
placed into 5m1 tubes containing 2m1 of a neutralizer solution comprising 6%
Tween TM
80 plus 1% sodium thiosulfate plus 5% bovine serum plus 10% Bovine Serum
Albumin.
[162] The tubes were sonicated for 20 minutes and then vortexed for 2
minutes.
Serial 10-fold dilutions were then made and and 100u1 of neat, 10-1 ,10-2 ,10-
3 and 10-4
dilutions were plated on Horse Blood Agar plates. The plates were incubated at
37 C
overnight and then enumerated.
[163] Control coupons, not exposed to disinfectant were similarly worked up
to
allow the log reductions to be calculated
[164] As can be seen in Table 20, the disinfectant according to Example 9
gave the
largest log reduction of biofilm.
Table 20
Log reduction Neutraliser control
Example 9 6.556 0.0437
Chlorclean TM (1000ppm 4.411 0.017
Cl)
SDIC (1000ppm Cl) 6.55 0.045
Example 16
[165] The bacterial reductions obtained from a 12-day dry surface biofilm
were
assessed under dirty conditions using Example 9, Chlorclean TM tablets and a
generic
SDIC tablet. Each test product was dissolved in artificial hard water
containing 340ppm
CaCO3 to which was added 5% Bovine Calf Serum.
Date Recue/Date Received 2021-09-08
34
[166] Coupons coated in 12-day dry surface biofilm were produced as per
Example
8. 2m1 of each test solution, followed by 2 ml of hard water to which was
added 5%
bovine calf serum were added to the wells in a tissue culture plate.
[167] After a 5 minute contact time, coupons were removed from the
disinfectant
solutions, rinsed twice using 30m1 phosphate buffered saline for 5 seconds,
and then
placed into 5m1 tubes containing 2m1 of a neutralizer solution comprising 6%
Tween TM
80 plus 1% sodium thiosulfate plus 5% bovine serum plus 10% Bovine Serum
Albumin.
[168] The tubes were sonicated for 20 minutes and then vortexed for 2
minutes.
Serial 10-fold dilutions were then made and and 100u1 of neat, 10-1 ,10-2 ,10-
3 and 10-4
dilutions were plated on Horse Blood Agar plates. The plates were incubated at
37 C
overnight and then enumerated.
[169] Control coupons, not exposed to disinfectant were similarly worked up
to
allow the log reductions to be calculated.
[170] As can be seen in Table 21, the disinfectant according to Example 9
gave a
log reduction essentially equivalent to that seen under clean conditions (see
Table 20).
It was also observed that both chlorine tablets gave essentially no log
reduction of
bacteria, suggesting complete neutralisation of the chlorine disinfectant by
the
proteinaceous soil.
Table 21
Log reduction Neutraliser control
Example 9 6.531 0.010
ChlorcleanTM (1000ppm 0.002 0.005
Cl)
SDIC (1000ppm CI) 0.007 0.018
Example 17
[171] In this example, the disinfectant according to Example 9 was tested
against
planktonic S. aureus, and compared to two commercially obtained oxidising
Date Recue/Date Received 2021-09-08
35
disinfectants, Chlorclean TM and OxivirTM Tb (Diversey Australia Pty Ltd,
Smithfield,
NSW, Australia), a ready to use solution comprising 0.5% hydrogen peroxide,
formulated with other proprietary ingredients.
[172] Alongside these commercial products some generic equivalents were
also
tested. These comprised Proxitane TM (Solvay Interox, Botany, NSW, Australia),
an
equilibrium solution of hydrogen peroxide, acetic acid and Peracetic acid
containing
27% hydrogen peroxide, 7.5% acetic acid and 5% of peracetic acid, an
unformulated
SDIC tablet (Redox Chemicals, Minto, NSW Australia) that released 1000ppm
chlorine
on dissolution in 10 litres of water, and a 6% solution of hydrogen peroxide
(Gold Cross,
Biotech Pharmaceuticals Pty Ltd, Laverton North, Victoria, Australia). These
generic
products were selected to try to match the active ingredients in the
formulated product,
thus assess the role of the product formulation.
[173] Where applicable, the disinfectant products were diluted using
artificial hard
water prepared by dissolving 0.304g anhydrous CaCl2 and 0.065g anhydrous MgCl2
in
distilled water to make one litre of hard water.
[174] Table 22 shows the products tested, and the concentrations of active
materials in the test samples.
Table 22
Product Sample preparation Concentration of active
ingredients
Example 9 8.5g powder dissolved in 1100ppm hydrogen peroxide
500m1 hard water 2200ppm PAA
ChlorcleanTM 1 tablet dissolved in 1 litre 1000ppm chlorine
hard water
Oxivir TM Tb Used undiluted 0.5% (5000ppm)
Accelerated hydrogen
peroxide
Generic equivalents
Proxitane TM 4m1Proxitane TM diluted to 10,080ppm hydrogen
100mlwith hard water peroxide
Date Recue/Date Received 2021-09-08
36
2,200ppm PAA
20g SDIC tablets 1 tablet dissolved in 10 litres 1000ppm chlorine
hard water
6% hydrogen 10m1 diluted to 100m1 with 0.6% (6000ppm) hydrogen
peroxide hard water peroxide
[175] Disinfectant efficacy in the absence of soil was tested by mixing 1
ml of test
disinfectant with 1 ml of hard water and immediately adding 10 1 of Tryptone
Soy Broth
(TSB) containing approximately 109 planktonic bacteria for 5 minutes contact
time. 1 ml
neutralizer (1% Na-thiosulphate, 6 % Tween TM 80, 5% BCS and 10% BSA in PBS)
was
then added.
[176] Disinfectant efficacy in the presence of soil was tested by mixing 1
ml of test
disinfectant with 1 ml of 5% bovine calf serum in hard water and immediately
adding
10[11 of Tryptone Soy Broth (TSB) containing approximately 109 planktonic
bacteria for 5
minutes contact time for 5 minutes contact time prior to the addition of 1 ml
of
neutralizer. 1 ml neutralizer (1% Na-thiosulphate, 6 % Tween TM 80, 5% BCS and
10%
BSA in PBS) was then added.
[177] Testing of these disinfectant systems against planktonic S. aureus
showed
that each one was capable of achieving a 7 logio reduction in the absence of
organic
soil. However, when tested under dirty conditions, only Example 9 retained its
full
efficacy.
[178] As can be seen in Figure 8, the presence of the organic soil
completely
deactivated the two chlorine-based disinfectants and the hydrogen peroxide.
OxivirTM
Tb however did exhibit some activity (0.67 logio). It is noted that in this
study OxivirTM
Tb was tested with a contact time of 5 minutes, whereas its manufacturers
recommendations are for a 10-minute contact time with bacteria.
Example 18
Date Recue/Date Received 2021-09-08
37
[179] The efficacy of the test disinfectants shown in Table 22 to kill the
organisms
within a dry surface biofilm of S. aureus was determined in the presence and
absence
of biological soil. Each condition was tested with five replicates for
determining residual
bacterial number (colony forming units ¨ CFU) using a 5-minute contact time.
[180] Disinfectant efficacy in the absence of soil was tested by mixing 1
ml of test
disinfectant with 1 ml of hard water and immediately adding a biofilm coated
coupon for
minutes contact time. 1 ml neutralizer (1% Na-thiosulphate, 6 % Tween TM 80,
5%
BCS and 10% BSA in PBS) was then added.
[181] Disinfectant efficacy in the presence of soil was tested by mixing 1
ml of test
disinfectant with 1 ml of 5% bovine calf serum in hard water and immediately
adding a
biofilm coated coupon for 5 minutes contact time prior to the addition of 1 ml
of
neutralizer.
[182] Positive (biofilm covered coupons) and negative (clean sterile
coupons)
control were subjected to the same treatments as described above but test
disinfectants
were replaced with hard water. Confirmation that disinfectant activity was
completely
inactivated by the addition of 1 ml of neutraliser was tested by adding 1 ml
of neutraliser
to test mixture (1 ml disinfectant plus 1 ml of either soil or hard water),
immediately
adding a biofilm covered coupon and reacting for 5 minutes (results not
shown).
[183] Determination of residual biofilm viability was determined by
subjecting
control and test coupons to sonication at 80 kHz for 20 minutes prior to
serial 10-fold
dilution and overnight plate culture at 37 C and CFU determination.
Results
[184] Positive control coupons had a mean of 2.6 x106 CFU/ coupon.
[185] In the absence of biological soil, and with a five-minute contact
time, example
9 was observed to give a 6.42 logio reduction, whilst the diluted Proxitane TM
sample
gave only a 2.04 logio reduction. The chlorine-based disinfectants, SDIC and
Chlorclean TM reduced biofilm viability by 2.85 logio and 2.82 logio
respectively. OxivirTM
was found to give approximately a 1 logio reduction whereas the unformulated
hydrogen
peroxide gave essentially a zero logio reduction under both clean and dirty
conditions.
Date Recue/Date Received 2021-09-08
38
[186] Under dirty conditions (i.e., in the presence of an organic soil),
Example 9
again gave a 6.42 logio reduction. Both SDIC and Chlorclean TM disinfectant
efficacy
was significantly decreased in the presence of soil, giving logio reductions
of 0.03 and
0.02 respectively. OxivirTM Tb also gave a reduced efficacy, giving a 0.24
logio
reduction of biofilm viability (See Figure 9).
Conclusions
[187] The disinfectant solution according to Example 9, along with two
other
formulated, commercially available disinfectant systems, each of which
contained an
oxidising biocide, along with other ingredients such as surfactants. The
effect of addition
of the proprietary ingredients to disinfectant efficacy was evaluated by
comparing the
formulated disinfectants with generic equivalents in a bid to determine if
biofilm removal
is due to the active ingredient alone or if proprietary ingredients act in
synergism with
the active ingredient.
[188] The outstanding performer in this study was Example 9 which
completely
inactivated the Dry Surface Biofilm in the presence or absence of soil.
[189] The formulated chlorine-based product Chlorclean TM , as well as
unformulated SIDC tablets were the next best performers, although they killed
significantly less biofilm bacteria (3 Logio) than Example 9, and only in the
absence of
soil.
[190] Previous studies have demonstrated that, chemicals such as
hypochlorite are
consumed by the surface layers of the biofilm causing depletion of the
neutralizing
capacity before the disinfectant can penetrate into deeper layers (see
reference 13)
making traditional hydrated biofilm more tolerant than planktonic cells to
these
disinfectants. However, a study on the efficacy of hypochlorite against Dry
Surface
Biofilm found that this semi-dehydrated biofilm was more tolerant to
hypochlorite than
traditional hydrated biofilm (see reference 6).
[191] Even in the absence of soil, the hydrogen peroxide-based
disinfectants killed
significantly less biofilm bacteria than disinfectants based on chlorine or a
combination
of peracetic acid and hydrogen peroxide. OxivirTM Tb killed approximated 1
Logio of the
biofilm bacteria while hydrogen peroxide solution had no effect. It is noted
however that
Date Recue/Date Received 2021-09-08
39
OxivirTm's manufacturer's recommended contact time for killing bacteria is 10
not five
minutes as used in the study and this could explain its lower performance.
However,
even a contact time of 5 minutes is probably excessive given the way that dry
hospital
surfaces are cleaned. The majority of disinfectants have no residual effect
and remain
active only when wet.
[192] The differences in kill rate between Example 9 (formulated additives)
and
diluted Proxitane TM (no additives) suggests that the activity of Example 9
against DSB
may be governed not only by the active ingredients (hydrogen peroxide and
peracetic
acid), but also by other factors such as the added surfactants or excipients,
chelating
agents or its solution pH.
[193] Surfactants may increase diffusion of the active ingredients into the
biofilm
(due to a lowering of the solution surface tension, and hence improved wetting
of the
biofilm surface).
[194] Increased diffusion is likely to result in increased biofilm kill as
all these
tested disinfectants, in the absence of organic soil, can kill 7 Logio of
planktonic
organisms. Chelating agents complex any calcium and magnesium ions present in
the
hard water, plus any other interfering metals often present in tap water such
as iron,
manganese and thus increase disinfectant performance in hard water.
[195] References
1. Vickery K, Deva A, Jacombs A, Allan J, Valente P, Gosbell IB; "Presence
of
biofilm containing viable multiresistant organisms despite terminal cleaning
on clinical surfaces in an intensive care unit"; Journal of Hospital
Infection,
(2012) 80, 52-55
2. Hu H, Johani K, Gosbell IB, Jacombs AS, Almatroudi A, Whiteley GS, Deva
AK, Jensen S, Vickery K; "Intensive care unit environmental surfaces are
contaminated by multidrug-resistant bacteria in biofilms: combined
Date Recue/Date Received 2021-09-08
CA 03082443 2020-05-12
WO 2019/097293 PCT/IB2018/001437
results of conventional culture, pyrosequencing, scanning electron
microscopy, and confocal laser microscopy"; Journal of Hospital Infection.
(2015) 91, 35-44
5 3 Whiteley GS, Knight JL, Derry OW, Jensen SO, Vickery K, Gosbell IB;
A
pilot study into locating the bad bugs in a busy intensive care unit"
American Journal of Infection Control, (2015) 43, 1270-1275
4. Almatroudi A, Hu H, Deva A, Gosbell IB, Jacombs A, Jensen SO, Whiteley
10 G, Glasbey T, Vickery K; "A new dry surface biofilm model: An
essential
tool for efficacy testing of hospital decontamination procedures"; (2015),
117, 171-176
5. "Standard test method for quantification of Pseudomonas aeruginosa
15 biofilm grown with high shear and continuous flow using CDC biofilm
reactor". ASTM E2562-12. ASTM International, West Conshohocken
6. Almatroudi A, Gosbell IB, Hu H, Jensen SO, Espedido BA, Tahir S,
Glasbey TO, Legge P, Whiteley G, Deva A, Vickery K. "Staphylococcus
20 aureus dry-surface biofilms are not killed by sodium hypochlorite:
implications for infection control"; Journal of Hospital Infection, (2016),
93,
263-270
7. Almatroudi A, Tahir S, Hu H, Chowdhury D, Gosbell IB, Jensen SO,
25 Whiteley GS, Deva AK, Glasbey T, Vickery K.; "Staphylococcus aureus
dry
surface biofilms are more resistant to heat treatment than traditional
hydrated biofilms", Journal of Hospital Infection (2018), 98, 161-167
8. Vickery K, Pajkos A, Cossart Y.; "Removal of biofilm from endoscopes:
30 Evaluation of detergent efficiency"; American Journal of Infection
Control
(2004), 32,170-176
9. Vickery K, Ngo QD, Zou J, Cossart YE.; "The effect of multiple cycles of
contamination, detergent washing, and disinfection on the development of
CA 03082443 2020-05-12
WO 2019/097293 PCT/IB2018/001437
41
biofilm in endoscope tubing"; American Journal of Infection Control (2009),
37, 470-475
10. Ren W, Sheng X, Huang X, Zhi F, Cai W. "Evaluation of detergents and
contact time on biofilm removal from flexible endoscopes"; American
Journal of Infection Control (2013), 41, e89-e92
11. Ying Fang, Zhe Shen, Lan Li, Yong Cao, Li-Ying Gu, Qing Cu, Xiao-Qi
Zhong, Chao-Hui Yu, and You-Ming Li "A study of the efficacy of bacterial
biofilm cleanout for gastrointestinal endoscopes" World Journal of
Gastroenterology (2010), 16, 1019-1024
12. Goeres DM, Loetterle LR, Hamilton MA, Murga R, Kirby DW, Donlan RM.;
"Statistical assessment of a laboratory method for growing biofilms'";
Microbiology (2005) 151, 757-762
13. Chen X, PS Stewart. "Chlorine penetration into artificial biofilm is
limited by
a reaction-diffusion interaction". Environ Sci Technol 1996;30: 2078-83
