Note: Descriptions are shown in the official language in which they were submitted.
WO 94/02022 ~ 1 ~ 0 8 9 ~ PCI /GB93/01478 I,
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Title: Im~rovements in or Relatinq to Germicidal~
,: ,
Compositions ;
, . ~
Field of the Invention
.
This invention relates to germicidal compositions, ,~
particularly` for use on surfaces, ie compositions capable `~
of destroying or inactivating micro-organisms,
particularly surface-bound micro-organisms.
Summary of the Invention -`~
. .
In its broadest aspect the present invention provides a `
surface germicidal composition comprising a dyestuff which
is capable of photo-dynamic inactivation of micro- 9'''~,``.'
organisms. ~
!:
It is preferred to use a dyestuff that generates singlet
oxygen on exposure to light.~ On absorption of light ~ ~-
energy a dye molecule is converted to a more energetic or ~;~
exited state ~S1 ) from its electronic ground state (SO). , -
(Electron spins are paired and SQ these are singlet states ! ` `
in the language of spectroscopy, having a single energy
level in a magnetic field.)
,L :`. .
;`."'.
The excited state is short lived and can lose energy and
return to the ground state in a number of ways: by
emission of a quantum of light as fluoresecence; by
internal conversion as the energy is degraded ~o heat; by
collision with a molecule of a different substance , !; `
if. . :`
. .
`. :.`.
W094/02022 PCT/GB93/014781 ~
Q,~?,g6 ~
(fluorescence quenching). `
. ~
The shor~ lived singlet s~ate may also undergo a pr~cess
called intersys~em crossing to a longer-lived excited
state, the triplet state. (The state is termed "tripletn
because the electron in the higher energy level is no
longer spin-paired with the electro~uin the lower level ~`
and the excited state has three energy levels in a
magnetic field.) ;~
`~
Interaction of the excited triplet state with ground state
molecular oxygen (which exists in the tripLet state
normally) regenerates the dye ground state as energy is
transferred to the oxygen which is promoted to the
.
electronically excited singlet state. This means that, ~`
under idea~l circumstances, a single photosensitiser
molecule can generate many times~its own concentration of -~
singlet oxygen.
Singlet~oxyg~en is highly reactive~and~photosensitised
oxidation~proceedlng via this route is known as Type II
photo-o~xida~t~ion. ~Type II photo-o~xidation is independent
of the~photosensit~iser used ~o gene~rate the s~ingIet 1~
oxygen.; ~An~important féature of the sensitiser is that it ~^
shoul~d have~a h~igh~quantum~yie}d~of triplet formation
(that is~ ideally;,~a triplet state~should be produced for ~`
ea~ch~photon absorbed~ Intersystém crossing to the ~-
~triplet~state~; i8~ ~facil~itated~by the presence of heavy
atoms in the molecule. `~
.
Photo-oxidation of any vital component of an organism may . ``
result in ce}l de~ath (protein, polypeptide, amino-acids,
lipids with allylic hydrogens, tocopherols, sugars and
cellulose).
, :~
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W094/02022 PCT/GB93/01478
2 i ~0896
3 --
Currently preferred dys~uffs include Rose Bengal (Ac;d Red
94, Colour Index No. 4S440), Ery~hrosin B (Acid Red 51,
Colour Index No. 45430), and ph~halocyanin sulphonates
such as aluminium phthalocyanin sulphonate (APS) and zinc
pht~. locyanin sulphonate (ZPS). Rose ~engal and
Erythrosin B are known food colourants (Rose Bengal is
Food Colour Red No 105 and Ery~hrosin B is Food Colour Red -`
No 14), and Erythrosin B is on an EEC list of colouring
agents allowed for use in cosmetic products, so these two ~
dyes are well suited for use in compositions intended for `;
domestic use. Mixtures of dyes can be used, and in some
cases it may be desirable ~o include in a mixture a dye ~
that will remain visible a~ the end of the photodynamic ~^;
process~
The concentration of dyestuff in the composition is not
critical and will typically be up to lOOppm, with good
results having been obtained with concentrations in the i~
~range lOppm to 20ppm. Lower concentrations, down to lppm
should~ also give reasonable results. ~"
Singlet oxygen has a short lifetime and therefore a short
pathlength for diffusion, 50 to be effective a ir;
photosensitising dye generating singlet oxygen must be };~
close to the target substrate. Preferred dyestuffs are ~-~
therefore substantive to (ie capable of binding to) micro-
. .
organisms, typically by binding to cellular protein on the
organism surface or other cellu}ar components~ eg cellular
fats. `-
'...
The preferred dyes men~ioned above generate singlet oxygen
on exposure to light and are substantive tO protein and so
capable of binding to micro-organisms via cellular
pro~ein. In this way, target~ed killing of organisms and
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W094/02022 PCT/GB93/01478~ ~
21~0896
hence germicidal action is possible.
It is also preferred to use a dyestuff that is bleached by ~
exposure to light. By use of a photo-bleaching dyestuff ;
that is substantive to miCro-Qrganisms itican be Qossible
for a visible indication of the pres~nce of micro-
organisms to be provided. As the dyestuff bleaches, the i
photo-dynamic action proceeds causing the death of, or `~
otherwise inactivating, micro-organisms. In the presence
of low levels of light, both bleaching and the photo- ~
dynamic activity are believed to proceed more slowly, ;
whereas at higher light intensities both processes occur
more quickly. Thus, depending on the relative rates o~
bleaching and photo-dynamic activity, the presence of -
visible dyestuff indicates to the user that the photo- -
dynamic inactivation of any micro-organisms present is -
incomplete. i
~ ~ .
The photo-dynamic inactivation of micro-organisms in
suspension by dyes such as Rose Bengal is known. See, for
example, Journal of Applied Bacteriology 1985, ~58] pages
391-400/ Photochemistry and Photobiology 1988, 148] pages
607-612 (Neckers et al), and Shokuhin Eiseigaku Zasshi `~
1962, 10(5), pages 344-347. However, it has now been ;~-
found that suitable dyestuffs are capable of photo-dynamic
inactivation of micro-organisms on surfaces, and this is `~
the basis of the present invention. It is well-known that
micro-organisms are much more susceptible to biocides in
their planktonic or suspended state: they are much more
difficult to inactivate when attached to surf`aces, which
is their usual or preferred state. Micro-organisms will
normally be on surfaces in the form of "biofilms", that
is, embedded in a matrix of extracellular material. This
extracellular material may sometimes be referred to as -~
W094/02022 21 ~ 0 8 9 ~ PCT/GB93/01478 ~
"adhesin" in the literature. It is therefore no~ obvious
that a process which acts on micro-organisms in their
planktonic state would act on surface-bound organisms
without modification being required. Surface-bound mlcro-
organisms represent an important and substantial source of
contamination in domestic, institutional and industrial ~
environments, and the present invention can enable `~-
targetted germicidal action on such micro-organisms.
Compositions according to the present invention are
particularly suitable for use on hard domestic and
industrial surfaces such as glass, plastics, ceramic and
metal surfaces. In particularly, the compositions are ~'~
effective for use on surfaces which may harbour soils
having the potential for bacteriological contamination in
surface imperfections, joints and other relatively
confined regions.
The composition is preferably acidic, eg having a pH in
the range of 3 to 5, eg a pH of about 4, as acidic s
compositions are found to have substantially enhanced ;
effectiveness against Gram-negative (G-) micro-organisms `~
as~compared with neutral compositions. The effectiveness i`
against Gram-positive (G+) micro-organisms seems not to be
significantly affected by pH. The compositions are
conveniently made acidic by use of relative}y mild organic
acid, such as àcetic acid. ~ ;~
, ~ .
Neckers et al (above) review conflicting evidence that
penetration of the dye Rose Bengal itself through the cell
wall is essential for inactivation. They suggest that
their own results support the dye penetration hypothesis i-
primarily on the basis of differential inactivation of G
and G- species. The envelope of G- bacteria has an
W094/02022 PCT/GBg3/01478~ ~
2~l~o896
- 6 -
additional outer membrane composed substantially of
lipopolysaccharide which Necker et al regard as serving as
a barrier to potentially toxic substances. An
alternative interpretation could be that the ~moun~ of
protein exposed in the cell walls is very different ;`
between G+ and G- species, being g~eater for G+ than G-, `r'~'
with a binding affinity for the dye which varies with pH.
The concentration of dye bound to the cell wall would
therefore be a function of pH. This interpretation would
account for our observations of the variation in kill rate i;
with pH (but not the apparent resistance of the G+ species
B. subtilis and B. megaterium). -
. .
There is no difference between the endospore-forming -
species B. s~lbtilis and the nonspore-forming S. aureus in
their susceptibilities to photodynamia action by Rose
3engal in the absence of endospores. The apparent
differences shown in our studies are presumably due to the
~presence of endospores both for B. subtilis and B. `~
mégaterium~which are clearly more resistant to sing}et ``
oxyge~n than the bacteria themselves. The spores survive
exposure to Rose Bengal and light and subsequently l`~
germinate to give countable colonies. Spores are known to
be difficult to stain and presumably have no affinity for ~
Rose 8engal under any of the conditions used. There `
appears to be little documentation on the toxicity of --~
singlet oxygen to spores in the literature. From studies ;~
on the possibly less resistant conidia of Neurospora `~
~crassa (Photochem. Photobiol., 33, 349 (1981)),singlet ~:
oxygen does have potential in this respect.
The composition msy optionally include other ingredients
such as one or more surfactants (for cleaning purposes)
and/or one or more solvents.
- W094/02022 214 089 6 PCT/GB93/01478 ;~
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The surfactant is preferably alkoxylated, more preferably `
ethoxylated, eg being in the form of ethoxyla~ed al~cohols.
The alcohol preferably has between 4 and 15 carbon a~oms, 1.. `,.3~"
is of straight or branched chain configuration, and has an
HLB value (hydrophilic lipophilic balance) in the range 10
to 14, eg 12.
A wide range of suitable surfactants are commercially
available, one such material being the surfactant
available under the trade name Imbentin 91-~5, from Kolb,
which is a nonionic C9-11 alcohol ethoxylate, having an
average of 5 moles of ethylene oxide per mole of alcohol.
,. . ..
Primary ethoxy sulphates may also be used. `Y
:
Mixtures of surfactants may be used if desired.
The surfactant is preferably non-ionic or anionic, or a
mixture of both types. `
, ;. ~
Preferred anionic surfactants for this purpose include ~;`;
primary~alkyl sulphates (PAS), preferably sodium dodecyl ~
sulphate (SDS). Commercial mixtures containing a ~-
substantial proportion of dodecyl sulphate (eg Empicol LX) `
are espcially preferred. Dodecyl sulphate is a known -~
protein denaturant, is good for cleaning protein off ``
surfaces, and is biocidal.
~he composition is preferably substantially free of `~
cationic surfactant, but may include a minor amount of
cationic germicide.
- ,,
Surfactant preferably constitutes an amount in the range `~
. - ..
W094/02022 PCT/GB93/01478~
2140896 , '`'i'
0.05 to 2.5% by weight of the total weigh~ of the
composition, typically 0.5% to 1.5% by weight, eg 0.7% by
weight nonionic surfactant with an optional amount of up ~-
to 0.2~ by weight of anionic surfactant. ~
.~ ~ .
The solvent is preferably polar an~is preferably a ~--
straight or branched chain C2 to C~5 alcohol such as
ethanol, butanol, isopropanol (propan-2-ol) (IPA), N-
butoxy propan-2-ol (propylene qlycol n-butyl ether), 2-
butoxy ethanol (ethylene glycol monobutyl ether). IPA is
the currently preferred solvent.
Dihydric alcohol such as ethylene glycol, and water
miscible ethers such as dimethoxyethane may also be used. -~
`: `
Mixtures of solvents can be used if appropriate, eg
mi~xtures of ethanol and N-butoxy propan-2-ol.
Solvent is preferably present in an amount in the range 2 ~-~
to 20t by weight of the total weight of the composition.
: .
At least some of these solvents, eg ethanol, weaken the
cell walls of micro-organisms,~making them more permeable
and so more susceptible to penetration by singlet oxygen.
This has the effect of enhancing the micro-organism-
~illing effect of the dyestuff.
It is found that inclusion of surfactant can reduce the
photo-dynamic effect of dyestuffs (possibly by
solubilising ~he dyestuff and preventing adsorption on the
cell wall), and inclusion of solvent can also reduce the
photo-dynamic effect of dystuffs (possibly by competing
for singlet oxygen). However, it is found that with three ` ;
component formulations, comprising dyestuff, surfactant
``;-.
'~,.
: . .
:` `
W094/02022 PCT/GB93/01478 ~
21ilO896 ~'
i,
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and solvent, the reduction in photo-dynamic effect of the j;:
dyestuff is less than would be expected ~rom the combined
effects of surfactant and solvent. The surfac~ant and 1`~
solvent together thus have a synergistic effect, the '~A
result of which is to lower the reduction in the photo-
dynamic effect of the dyestuff.
In a preferred aspect, the present invention thus provides `i
a surface cleaning and germicidal composition, comprising
a dyestuff which is capable of photo-dynamic lnactivation
of micro-organisms, a surfactant and a solvent.
The composition may include a number of optional
ingredients including ~he following: ~
1. Detergent boosters, preferably metal chelating agents --
such as ethylene diamine tetra acetic acid (EDTA). Metal
chelating agents (including EDTA) have also been claimèd
to permeabilise cell walls, thus making organisms more
susceptible to biocides.
. ..
~;
2. Electrolyte such as a buffer or salt, eg Na2S04, which
acts to assist binding of dye to protein by promoting ~`
movement of dye from the aqueous~ phase to the protein
salt. -Electrolyte is commonly present in various dye :~
formulations as commercially available, although -~
~additional electrolyte~can be added if required. Total `~
electrolyte content of the composition would~ypically be
in the range 0 to 1~ by weight, preferably about 0.1~.
3. Perfumes.
4. Thickeners. ``
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W094/02022 PCT/GB93/01478~
2~40896
1 ~ _ ;
The composition is in the form of an isotropic, single
phase composition and is of particular use as a germicide
(possibly also with a cleaning effec~) on hard surfaces, `
finding application in a wide range of contexts, including
domestic applications, eg kitchen and-bathroom surfaces -
including toilet bow~s, in institu~tions such as schools,
hospitals etc, and in commercial premises such as
factories, offices, hotels etc.
:..
For domestic use at least, the composition is preferably
formulated as a product intended for application by
spraying and is conveniently packaged in a suitable
container, eg having a hand operated trigger spray or an ;
aerosol propellant dispenser. The container is preferably `
light-opaque. ,~-
, . ~.
In use, the composition is applied to a surface to be
treated in any convenient manner, eg by spraying from a 1;~
suitable dispenser, wipinq on with a carrier such as a
cloth or sponge, or pouring from a container etc. This
might in some cases, particularly in industrial cleaning,
be followed by exposure to a light source, eg a white ~;
light source such as a quartz halogen lamp or fluorescent ;~
"daylight~ source. (The process would be an alternative
to using danqerous germicidal radiation, for example from
a low pressure mercury discharge lamp emitting resonance ~--
radiation at 254 nm. Such radiation is harmful to the
unprotected eye.) This would genérally be followed by a `
rinsing step, if required, eg by wiping with a carrier,
application of a stream of running water etc.
In a further aspect, the invention thus provides a method
of killing bacteria on a surface, comprising applying to
the surface a composition in accordance with the
~ ,:
W094/02022 PCT/GB93/01478 s:
21~0896 ~
invention.
The invention will be further described, by way of
illus~ration, in the following Examples and by reference
tO the accompanying Figures in which:`~
` ,s`
Figure 1 shows two bar charts of log (reduction) values`-i
illustrating the lethal effect of Rose Bengal and light on ;-~
various micro-organisms in suspension at pH4 and pH7, with
Figure la showing results for Gram-positive micro- .-
organisms, and Figure lb showing results for Gram-negative `
organisms and yeasts;
.. .
Figure 2 is a pair of graphs of log (reduction) versus pH
showing the biocidal effect of Rose Bengal and light on S. `
aureus and E. coli as a function of pH, with Figure 2a.~.
showing results after 20 minutes expo~ure to light and
Figure 2b showing results after 60 minutes exposure; -
Figure 3 is a pair of graphs similar to Figure 2 obtained
using Erythrosin B in place of Rose Bengal; :~
j~
Figure 4 shows two bar charts of ?9 ( reduction) r ~,'
illustrating the photohygiene effect of various
combinations of Rose Bengal (RB), Imbentin C91-35 (AE),.
isopropranol (IPA) and Empicol LX (PAS), with Figure 4a
showing results obtained without exposure to light andL`
Figure 4b showing results obtained with èxposure to
light; ```:
. :~
Figure 5 is a graph of adsorption of Rose Bengal by E..
coli, with amount adsorbed (nanomoles) versus equilibrium ::
concentration (micromoles/l), with results at pH 4 shown ~.
by squares and results at pH 7 shown by crosses; ':.
:
W094/02022 PCT/GB93/01478~ `
21~0896 ' ,.".,.
- 12 -
FiguLe 6 is a pair of graphs similar to Figure 5 showing ;.~
the effect of electrolyte, with Figure 6a showing results ;
at pH 4 and Figure 6b showing results at pH 7, with
results without additive shown by squares, results with
sodium sulphate (1%) shown by crosses.and results with :
sodium sulphate (5%) shown by double~;.crosses; ~.:
Figure 7 is a pair of graphs similar to Figure 5 showing
the effect of surfactant, with Figure 7a showing results
at pH 4 and Figure 7b showing results at pH 7, with .
results without additive shown by squares, results with ~-
non-ionic surfactant (0.7%) (NI) shown by crosses and -~;
results with PAS (0.7%) shown by double crosses; .~:
Figure 8 is a graph similar to Figure 5 showing the effect
of solvent at pH 4, with results without additive shown by
sma}l squares, those for 10% IPA shown by crosses, those ~.
for 0.7~ Imbentin shown by double crosses and those for .`
10% IPA and 0.7% Imbentin shown by large squares;
Figure~9 is~ a qraph of log (reduction) versus exposure
:time~(minutes) showing the effectof pH on thie rate of kill ~.
of E. coli by Rose Beng:al, with results at pH 4 shown by
. -
squares and results at pH 7 shown by crosses; and .-
: Figure 10 is a graph s:imilar to Figure 9 showing the .`
: effect of electrolyte at pH 7 on the rate of kill of .-:
E.coli by Rose Bengal,:wi~th r.esults without electrolyte:i`
shown by squares and r~esults with Na sulphate shown by `.
crosses. ; .
',`':~''''.
. Examples .~..
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W094/02022 21~ 0 8 9 6 PCT/GB93/01478 l;~
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EXPERIMENTAL PROCEDURES -~
~ ` .
Preparation of Inocula ~ .~
... .
~.
The following micro-organisms (generally either fro... he !'''"
National Collection of Type Cultures (NCTC) or the
American Type Culture Collection (ATCC~) were used in the
experiments described:
.,'`-''-
Bacteria: ~:
,~."~,.
Staphylococcus aureus NCTC 6538 (Gram positive)
Escherichia coli NCTC 8196 (Gram negative) "
~;
Pseudomonas aeruginosa NCTC 5940 (Gram negative) ~.
Enterobacter sp. NCTC 3281 (Gram negative) rr~.
Klebsiella sp. ATCC 11677 (Gram negative)
Bacillus subtilis .NCTC 6432 (Gram positive)
Bacillus megaterium NCTC 7581 (Gram positive) ;~.
Yeast:
Candida albicans j
., ~ .
Organisms were grown up by overnight incubation in . .
: nutrient broth at 37-C for bacteria (28-C for Ps. .-
. .
aeruginosa) or SABS broth (SABS is Sabourand Dextrose
Agar, with liquid medium in the case of SABS broth, from
Oxoid ~td) at 28-C for yeast. Cultures were isolated by
vacuum filtration using a 0.45um Millipore fi}ter and
washed with quarter-strength Ringers solution before
resuspension in Ringers solutio~n (lOml). The organisms in `~
suspension were enumerated by serial ailution and plating
with nutrient aqar (bacteria) or SABS agar (yeast) and the .`
total viable count (TVC) expressed as the decadic !,~,~
- W094/02022 4089 6 PCT/GB93/01478l
logarithm of the number of colony-forming units (cfu) per -~
ml.
..
Experiments were carried out at pH 7 unless otherwise
specified. -~`
1 ) AGAR DIFFUSION DISC METHOD `~
Aqueous solutions containing 100 ppm of dye were prepared.
Aliquots (1Oml) of each dye solution were ste;rilized in
glass universal screw cap vials. Antibiotic assay discs
(13mm from BDH) were also sterilized. All organisms were `
grown overnight in nutrient or SABS broth (10ml).
For each micro-organism, two nutrient agar plates ( SABS "
agar for the yeast) were seeded with the overnight culture `
t10ul) to give confluent growth over the whole plate. -
~Using aseptic techniques, an antibiotic disc was dipped `~
into the first dye solution and placed on the surface of a
seeded agar plate. This was repeated with two other dye
solutions to give three discs on duplicate plates. -
One of each pair of duplicate plates was immediately
placed in an incubator at the appropriate temperature with
m~inimal exposure to light. The remaining plates were
placed on top of a light box for 3 hours. Illumination `;
from the light box was diffused white light of mean
intensity 4000 lux from fluorescent "daylight" tubes (2 x
15 watt, Exal X-ray Accessories Ltd, Hemel Hempstead). -~
Light intensities were measured at the surface of the ``
diffuser using a Megatron DA10 light meter. After ~`
exposure the plates were incubated overnight and then
examined for zones of inhibition around the disc.
,-~
- W O 94/0202~ 2 ~ 4 08g 6 P ~ /GB93/01478
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- 15
U~ing this approach, the following results weL-e obtained:
:
Example 1 (Disc Method)
, ~.
Results on agar for aqueous dye solutions of Rose Bengal,
Erythrosin B and aluminium phthalocyanin sulphonate (APS) -
(100 ppm) at pH 7 after exposure to light for 180 minutes ,::
as described above are summarised in Table 1. In the
Table, the results are expressed as the difference (in .
millimetres) between the radius of ~he clear zone of
inhibition (the area of no bacterial growth on the spread
agar plate) and the radius of the disc. Thus, the higher
the value, the greater the baterial kill. .
The agar diffusion disc method ranked Rose Bengal as more ,:`
efective than the structurally similar Erythrosin B. It
is tempting to ascribe this ranking to a difference in the
quantum yield for singlet oxygen formation. In methanol,
the quantum yield for singlet oxygen formation is 0.76 for ~
Rose Bengal compared to 0.6 for Erythrosin B. However, a .number of other factors might also be expected to ~-
contribute to the observed differences such as the rate of `ii
dye diffusion or differences in dye binding to the agar
gel or disc material. ~`' . .
A particular feature of the results from the disc method .
is the clear distinction in the sensitivity of Gram~
positive (G+) and Gram-negative (G-) organisms to
photodynamic action, at least at pH 7. Possible reasons -~for the relative resistance of G- organisms are discussed ~.`
elsewhere. ~`
F ~;
. 2) SURFACE TEST ~ `
~`'`.`.
~-~
"'~
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W094/02022 PCT/GB93/01478i ~
2~0~96
- 16 -
Test Solutions
Rose sengal (20ppm) in pH 4 b,ùffer and: `
.. `~ ' ~;'
1. No further addition ~
2. Ethanol (10 ~ v/v) and Imbentin C91-35 (0.7%). `
3. Propan-2-ol (10% v/v) and Imbentin C91-35 (0.7%). ~-
Sodium hypochlorite solution (0.125 %) was used as a
positive control.
Estimation of Number of Organisms Adherent to Base of -~
Petri Dish
,.,
Micro-organism, eg S. aureus, suspension (0.5ml) was added
to aliquots (100 ml) of quarter-strength Ringers solution
and the average cfu per ml determined (TVC). Aliquots ~`
(20ml) of these solutions were pipetted into sterile petri `
dishes and left at room temperature for 5 hours. The
inoculum was then removed by pipette into a sterile bottle
and the average cfu per ml remaining in suspension
es~imated. The number of organisms (as cfu) per square cm
adherent to the Petri dis~h was calculated from the
differencè in the solution concentrations.
.
Test Method
~.
Bacterial suspensions in quarter-strength Ringers ~20mls) ~;
were pipetted into sterile plastic Petri dishes which were ;~`~
left at ambient temperature for 5 hours. The inoculum was
then removed, the dishes washed once with Ringers solution ~
with gentle swirling by hand, and the solution poured off. ~;`-
Pairs of bacterially contaminated plates were treated with ~`
W094/02022 21 ~ n 8 9 6 PCT/GB93/01478
- 17 -
the test solutions. An aliquot (20ml) of ~est solution
was poured into one dish and ~he solution decanted after
30 seconds. The dish was rinsed with pH 4 buffer and
placed on the light box for 20 mins. In a separate /~
experiment, the solution was exposed in the second dish on -~
the light box fol- 20 minutes before being decanted and the
dish rinsed with pH 4 buffer. Both experiments weLe `
duplicated. -
,.. ...
After light exposure, one of ~he duplica~e plates was
overlaid with Tryptone Soya agar containing 1% glucose and
0.015~ Neutral Red cooled to abou~ 50C. The other ```~
duplicate plate was stained with 0.01% Acridine Orange for ~;
30 seconds, rinsed and examined microscopically (Nikon
"Optiphot" microscope equipped with a 100x apochromat oil- ~;
immersion objective, 10x eyepiece and epifluorescence ;;~-`
attachment with a B2-A combined filter/dichroic mirror / ~`~
block and super high pressure mercury lamp). The overlaid ~-
agar plates were incubated at 37C for 48 hours, by which
time colonies had grown out of the adherent bacteria which
had not been killed. The colonies took up the ~eutral Red
and could be seen and counted under the agar on the
surface of the dish. (A M R MacKenzie and R L Rivera-
Calderon, Agar Overlay Method to Measure Adherence of
Staphylococcus epidermidis to Four Plastic Surfaces, `~
App}ied and Environmental Microbiology, 50, 1322 (198S).)
Plates stained with Acridine orange were examined and
photographed. The number of stained bac~eria were counted
in a field of view (clumps counted as one) which had
previously been estimated by photographing a micrometer ~;
scale.
Example 2 (Surface Test) ``
W094/02022 PCT/G~93/01478f
~40S96 ' ~;
- 18 ~
Control experiment surfac~ tests using both direct
epifluorescence microscopy as described above and
suspension depletion gave similar values for the number of
bacteria (S. aureus) that could,~e attached to the surface
of a plastic dish (of the ord~r,~of one million per square ''
centimetre). '' , '''
., ~
Contracting the surface with the positive control (sodium ,~-,,
hypochlorite solution, 30 seconds) reduced the number of '~,
viable bacteria to zero. The results for photo~
dynamically inactivated bacteria are summarised in Table 2 '~
as the decadic logarithm of the reduction (log
(reduction)) of viable bacteria before and after exposure,
ie log (start count) - log (final count).
; ,~, . ` ',`
Example 3
. ., ~
Formulation: Rose Bengal (20 ppm), nonionic surfactant
(Imbentin C91-3S, 0.7%), propan-2-ol (10 %) (pH 4). ,i"
. . .
Experimental procedure was as previously described éxcept i~
that surface attachment of the~bacteria required them to
be in the exponential growth phase. This was achieved by
incubation in nutrient broth (for bacteria) or SABS broth
(for yeast) in the plastic Petri-dish for 3 hours. In '
Example 2, a suspension of non-growing bacteria in
Ringers' solution was simply allowed to stand for 5 hours. '~
This worked well for S. aureus but not other bacteria. `'`
,
The results are shown in Table 3. '~`"
'Previous experience using direct epifluorescence '~`
microscopy suggests that where confluent growth was `,''
obtained, this would be equivalent to an initial surface
'`''
.
"~
,
~.. W094/0202~ PCT/GB93/01478 ~
21~0896 `~`
- 19 - '~
density of viable microbes of the order of 1 million/sq.
cm. The sur~ace area of the Petri-dishes used was abou~
57 sq.cm. so tha~ the treatmen~ in all cases has reduced
the level of surface contamination by a~ leas~ 5 orders of
magnitude (log 5). ~;
, ~ ,,
Surface tests are more difficult to perform than ;
suspension tests, and for this reason mos~ experimental .-~
work has been performed in suspen~ion to demonstra~e the
effect on varying conditions.
.~,;
3) SUSPENSION TEST
Preparation of Solutions
Stock solutions of the following were prepared by weighing
and sterilized (except those containing solvent): :
Rose Bengal (0.2 percent) in propan-2-ol (95 percent)
Nonionic surfactant (Imbentin:C91-35, 14 percent)
(sometimes referred to by ~the abbreviation AE, for alcohol
ethoxylate)
Anionic surfactant (Empicol LX, 14 percent) (sometimes
referred to as PAS)
pH 4 buffer (citric acid (0.1 M,307 ml) + dibasic sodium
phosphate (0.2 M, 193 ml) :.
pH 7 bùffer (sodium dihydrogen orthophosphate (0.4M,468 ~:
ml) ~ disodium hydrogen orthophosphate dodecahydrate,
(0.4M,732 ml)
Buffers pH 5,6,8,9 were prepared as indicated in the CRC
Handbook of Chemistry and Physics, 8-36, 73rd Ed., CRC .
Press (1992-1993) '
Final concentrations in the test solutions were
' ~,
W094/02022 PCT/GB93/01478~
i -:
2~0~96 ~.
- 20 -
:
typically~
.. .
Rose Bengal - 20ppm
Ethanol - 10.0% (v/v) ~ ~-
Surfactant - 0.7% (w/v) ~
. ' ',~,.
In some examples, different concentrations were used as
specified. ``
", . ..
Test Method ~`
Test solutions were made up in sterile plastic petri
dishes to a depth of 5mm (30mls). A suspensian of micro-
organism tO.3ml) was added to each solution and gently
mixed in. If Rose Bengal was to be included in the test
solution it was added last to minimise light exposure.
Solutions were either exposed on a light box, placed in
the dark (conditions of reduced light exposure) or left on
the bench. The average intensity at the surface of the
light~box diffuser was 4000 lux measured with~a Megatron
DA 10 Iight meter tfrom Megatron Ltd). After specified `~
exposure times, surviving bacteria were enumerated as
colony-forming units (cfu/ml) following incubation after ~
serial dilution and plating onto agar. The decadic ;.~,
logarithm of the number of bacteria remaining (as colony- ;
forming units per ml) was determined and compared to the -~
number before exposure as log (start count) - log (final `~
count). The higher the value, the greater the bacterial `-
kill~ -
Suspension tests have been carried out against a variety ;
of organisms under a variety of conditions to optimise
photo-dynamic action against a range of micro-organisms,
including Gram-negative organisms and yeast. Salient '~
. `
`:
.
W094/02022 2 14 0 8 9 6 PCT/GB93/01478 i~;
~ .
- 21 -
results are summarised in the associated Tables. In these
Tables results are expressed as the decadic lagarithm of
the ratio of the initial number of colony-forming u~its
per ml to the number remaining after exposure, the log
(reduction~. Using this notàtion a value of zero means no
change in the number of organisms following exposure to
the conditions. The notation ~+" preceding a log
reduction figure indicates that no micro-organism growth ~5
could be observed (ie total kill).
Exam~le 4
Suspension tests were carried out as described above to
show the lethal effect of Rose Bengal and light on micro-
organisms in suspension; particularly the synergy of low
pH and ethanol solvent. Tests were carried using Rose
Bengal (20ppm) alone or with ethanol (1~ (v/v), with
light exposure of 20 minutes (light box). The results
expressed as log (reduction) values are qiven~in Table 4. ~-
Longer exposure times (of 60 and 100 minutes) improve `
performance for Gram negative organisms, part~icularly at
pH 7.
It will be seen that performance for Gram negative ~`
organisms is much improved at pH 4 as compared with pH 7.
~;
Example S ~
. .
Suspension tests were carried out as described above at pH
4 and pH 7 to show the lethal ef..~ct of Rose Bengal
(20ppm) and light (20 minutes exposure on a light box) of
various Gram-positive and Gram-negative micro-organisms `
and yeast in suspension, and the results expressed as log
:
W094/02022 PCT/GB93/01478 ' ~ ~
2~4~96
- 22 -
(reduction) are shown graphically in Figures la and 1b, ;
with Figure 1a giving results for Gram-positive micro- -~
organisms and Figure lb givin~ esults for Gram-negative A,.
micro-organisms and yeast. ~
... ...
... ..
Example 6 ,-
. .
Suspension tests were carried out as described above at a s;`
range Oe different pHs to show the biocidal efSect of Rose
Bengal ~20ppm) and light on S. aureus (G~) and E. coli (G-
) as a function of pH, and the results expressed as log
(reduction) are shown graphically in Figures 2a (exposure
time 20 minutes) and 2b (exposure time 60 minutes). In
the Figures, crosses show results for control (G-), double
crosses show results for E. coli, inverted triangles show
results for~control (G+) and triangles show results for S.
aureus~. X
Similar suspension tests were carr~ied out to show the `~
biocidal effect of Erythrosin B and light on S. aureus and `'
E. coli as~a function of pH, and the results are shown
graphically in Figures 3a and 3b, which are otherwise
identical to~Figures 2a and 2b. These show that
Erythr~osin~B performs similarly ~o Rose Behgal in terms of ``
its photobiocidal profile with p~
:
, .
Example 7
Further suspension tests were carried out as described ~
above at pH t using the following solutions: -
Nonionic surfactant Imbentin C91-35 (0.7%) `
bentin C91-35 ~0.7%) with Rose Bengal (100 ppm)
Anionic surfactant Empicol LX (0.7~
Empicol LX with Rose Bengal (100 ppm)
.~:
.,
W094/02022 2 14 0 8 9 6 PCT/GB93/01478
- 23 -
Imbenlin C91-35, Empicol LX (bo~h 0.7~) and Rose Bengal ;~
(lO0 ppm).
The resul~s are set OUt below in Table 5. In ~he Ta~le
~he term "dark" indica~es conditions of reduced light
light exposure rather ~han total darkness because of the
practical difficul~ies of avoiding some light exposure.
In the Table the term '~light~ indicates results that are
the average of several experiments carried OUt: over a
range of times (20 mins, 1 hour, 3 hours) in a
statistically designed experiment.
Results obtained in similar manner for E. coli at pH 7 are
shown graphically in the bar charts of Figure 4. In this '`~''i-~!'`'',
Figure PAS is used as an abbreviation for Empicol LX, IPA
is used as an abbreviation for isopropanol and AE as an
abbreviation for Imbentin C91-3S. This figure represents
averaged data for AE 0.7%, PAS 0.7%, IPA lO~.
`:
Example 8 ~
"`',','`
Further suspension tests were performed at pH 4 for E.
coli using one or more of the following: Rose Bengal
40ppm, surfactant 0.7% (Imbentin C91-35 or Empicol LX),
IPA (isopropanoI) 10~. Results are given in Table 6.
The following examples concern suspension tests carried
out generally as described above, but at pH 4. Rose
Bengal when used was present at a concentration of 20ppm,
although in some cases control solutions without Rose
Bengal were exposed to light and the results for these are
given in the column headed UNo Rose Bengal".
. ,~..
Examp es 9, 10, 11 and 12 used the Gram positive organism ~`
S. aureus, and Examples 13 and 14 the Gram negative
organism E. coli. Other reagents used are indicated in
`
W094t02022 PCT/GB93/01478
o~96 `~
- 24 -
the examples. In all these Examples, samples were exposed
for 20 minutes on a light box. The average intensity at
~he surface of the diffuser was~4000 lux measured w~lth a
Megatron DA10 light meter (~om Megatron Ltd).
.~ .. , ,. ~ .
Example 9 i
,~,.
Suspension tests were carried out using Rose Bengal,
ethanol and Imbentin C91-35, with S. aureus. The decadic ~
logarithm of the starting concentration, log (start), of -`;
S. aureus was 6.8. Results are given in Table 7. ;
:
Example 10
,~.
Suspension tests were carried out using Rose Bengal,
Dowanol PnB and Imbentin C91-35, with S. aureus. The log
(start) was 6.9. Results are given in Table 8. ;-
~ . .
This example shows that Dowanol PnB has certain biocidal ~:
properties. ~-
Example 11
Suspension tests were carried out using Rose Bengal,
ethylene glycol and Imbentin C91-35, with S. aureus. The ~-~
log (start) was 6.8. Results are given in Table 9. `~
.
Example 12 -
Suspension tests were carried out using Rose Bengal, IPA
and Lialet 111, with S. aureus. Lialet 111 is the trade ~;
name of an ether sulphate formulation commercially ¦~
available from Enichem, having an average chain length 11 '~
W O 94/02022 2 1~ 0 8 9 6 P(~r/GB93/01478 '~
!. ~,
- 25 -
with an average degree of ethoxylation of 3. The log
(start) was 6.7. Results are given in Table 10.
Example 13
Suspension tests were carried out using Rose Bengal,
propan-2-ol and Imbentin C91-35, with E. coli. The log ;~
(start) was 6.8. Results are given in Table 11.
-~.
Example 14
Suspension tests were carried out using Rose Bengal,
ethanol and Imbentin C91-35, with E. coli. The log
(start) was 7.1. Results are given in Table 12.
Example 15
Rose Bengal adsorption was detérmined from the depletion
in solution concentration. Concentrations were obtained
spectroscopically from absorbances measured at the
wavelength of maximum absorbance (ca. 549 nm) using a WPA
Linton S110 spectrophotometer on supernatant liquors freed
from microbes by centifugation. -~
';~
The results ar shown in Figures 5 to 8. ~;
The results show that the photobiocidal effect is ~-dependent on dye adsorption. Dye adsoprtion is:
a) increased by low pH (Figure 4); ~`~
b) increased by netural electrolyte (which also
increases the photobiocidal effect at neutral pH ~-~
on E. coli) (Figure 6); -~
:, .
c) decreased by surfactant (Figure 7); ~
W094/OZ02~ 96 PCT/GB93/01478
26 ,.. ! :;
d) increased by propan-2-ol (Figure 8).
: .
Calculations show the amou~t`;adsorbed onto E. coli ig of
~he right order of magnitude for monolayer coverage, given `~
that dyes are known to aggregate and the calculated 1?'`'
surface are of E. coli must be an underestimate (no ~`~
. .
account taken of fimbriae/pili). ~rief details of the ~-
calculations are given below. The molecular dimensions of `-
Rose Bengal were taken from a scale model (Catalin Ltd., ~`
~ondon). -
',
Surface area of E. Coli lE7 sq.nm
Area of Rose Bengal 2 sq.nm (flat) ~`
0.5 sq.nm (side)
~.
Monolayer coverage SE6 (flat) or 20E6 (side) ,'~
Measured adsorption 2 - 8 E-16 Moles/bacteria
Number of molecules 120 - 480 E6 per bacterium
Example 16 i"~
.,~,.
Suspension tests were carried out as described above to
determine the effect on the rate of kill of E. coli by `~
Rose Bengal (20 ppm) of pH and addition of electrolyte (Na
sulphate, 5~) at pH 7, and the results are shown
graphically in Figures 9 and 10, respectively. ~;
'~
~ .
` ~'`
W094/02022 21 4 0 8 9 6 PCT/GB93/01478
- 27 ~
''
.
Table 1 (Example 1) .
Photobiocidal Effect of Disclosing A~ents
Zone of Inhibition Around Dlsc
Gram
Organism Type Rose Bengal Erythrosin B APS
S. aureus .+ 4 1 2
B subtilis + 3 - 1 t
B meqaterium + 3 1 1.5 :
E coii - 0 0 0 :K Pneumoniae - 1.5 0 3 :~
Ps aeruqinosa - 0 0 0 ~
Enterobacter sp. - 0 0 0 ~.
C. albicans 0 0 0
.
.
Table 2 (Example 2)
~.:
~ .,
: The Lethal Effect of Rose Bengal and Light on
Staphylococcus Aureus Attached to a~P astic Surface
Solution Log ~Ratio)
Rose Bengal 4.7
Rose Bengal + - ~
Imbentin C91-35 + 6.0 . ~-
Ethanol
'
W094/02022 . PCT/GB93/01478( .. -;
" ~ ~o~96 _ 28 -;
:, : ',.
. .
Table 3 (Example 3)
Organism Experiment Results from Agar Overlay Technique ~i
Number Control After Exposure
S. aureus 1 Confluent growth No growth ~`
2 Confluent growth No growth
E. coli 1 Much growth 45 cfu `
2 Confluent growth No growth .
R. pneumoniae 1 Confluent growth No growth ~.
2 Confluent growth No growth i~.
P. aeruginosa 1 Much growth 5 cfu ~-
2 Conf~uent growth No growth
C albicans 1 Confluent growth 5 cfu . A
2 Confluent growth 4 cfu ~-~
,; , . .
, ' :!.:
.'~.'i'~';
~ Table 4 ~Example 4)
. ", ..~:
Organism Gram Type Conditions of Exposure .~i
No Solvent With Ethanol ~- .
~ pH4 ~ pH4 !'~
S~ aureus + 7.0 7.1 7.1 6.9
B subtilis + 0.6 2.1 3.1 0.8 ~1
B megaterium + 1.3 0.3 1.1 0.3 -:
E. coli - 0.2 5.3 0.1 6.9 ~;~
K. pneumoniae - 0.9 5.6 0 7.0 ~:
Ps. aeruqinosa - 0 7.0 0 6.9
Enterobacter sp. - 1.1 . 7.3 0 7.4 `-~`
C. aIbicans 0 5.7 0 5.7 ::.
Controls (No Rose Bengall
E. coli - 0.1 0.8 ~``
~ : .
:.,. : ,
.. . .
. ;`,`'
WO 94/02022 214 0 8 9 6 PCI/GB93/01478
'~ ~
.
-- 29 --
Table 5 (Example 7) :
,
ORGANISMIMBENTIN IMBENTIN/RB EMPICOL EMPICOL/RB ~:
light dark light dark light dark light dark
St. aureus 4.5 4.0 8.0 3.5 4.5 4.5 5.5 5.0 ~-~
: ` `.
E. coli 9.0 9.5 10.5 10.0 5.5 5.0 6.5 6.0 .
Entero 0 0 0 0 0 0 0 0
Klebsiell2 7.0 6.0 6.0 7.0 6.0 6.0 6.0 6.0
Ps. aerug. 0 0 0 0 0 0 0 0 ~'
~"
C. albicans 0 0 1.0 1.0 2.5 1.5 3.0 3.0 ~:
,~
-
~ ORGANISM IMBENTIN/EMPICOL IMBENTIN/EMPICOL/RB ROSE BENGAL
3 ~ lioht dark liaht dark : liqht dark
,:
St. aureus 3.0 4.0 2.0 0 4.0 0 ~`
E. coli 4.0 4.0 4.0 4-5
Entero. 0 0 0 : 0 0 0
,,
Klebsiella 3.5 3.5 4.0 2.5 0 0
. . .~
Ps. aerug. 0 0 0 0
~C. albicans 0 0 0 0 0 0
---
~ .
`:
.~
W094/02022 PCT/GB93/01478~
~,~4~9 .~
- 30 - ~
, ~ ;,
~:,
.. ~ . ~ .
Table 6 (Example 8) ; ,.
.
~ ,.
EXPOSURE TIME
1 HOUR 2:HOURS 3 HOURS
light dark light dark light dark
Rose Bengal 3.8 0.5 7.2 0.4 7.2 0.7 .
RB/Imbentin
C91-35 0.2 0.2 1.0 0.3 3.8 0.5 `.-
RB/Empicol LX 1.7 0.4 3.9 0.7 7.2 0.7 ~ .
RB/IPA 5.4 3.6 4.7 5.0 7.2 7.2
'- ~ Imbentin
C91-35~ ~ 0.1 0.3 0.9
Empicol LX : 0.1 0.6 1.0
IPA 0.3 1.7 4.4
.
: : ,~
,
,,
. W094~02022 ~1 4 n 8 9 6 PCT/~B93/01478
- 31 - -:
Table_7 (Example 9)
,~
, ....
Log (reduction) ~:
Ethanol Imbentin After No
% C91-35 ~ Light Exposure Rose Bengal
0.2 +6.8
150 0 66 +6 8
0.6 +6.8
- +6.8 0.1 -
+6 r 8 ~ O ~ 4
- +6.8` 0.0
- 0.2 4.6 2.5
- 0.6 3.5 2.3 - ..
- - +6.8 2.3 - '.
,~,
.
Table 8 (Example 10)
- Log (reduction)
:~: Dowanol Imbentin ~fter No
; % C91-35 % Light Exposure Rose 8engal
:~ ~ 3: . :0.7 +6.9
3 - - :. +6.9 4.0 `~
- 0.7 5.2 3.4 `~
_ - +6.9
: ~.
~:
~.
WO94/02022 PCr/GB93/01478 (
2~40~96
!~ .
-- 32 --
-
Table 9 (Example 11) ~ "~
; .
,-~ ".
.. Log keduction) ~;
Ethylene Imbentin . ~ After No
Glycol % C91-35 %Light Ex-posure Rose Bengal
0-7 +6.8
~10 - +6.9 -0.2
- ~ 0.7 . +6.8 3.4
+5.8
~ble 1 0 (Example 12)
Log ( reduction) ~.
~Propan-2-ol Lialet 111 ~After No
% ~ ~ % Light Exposure Rose Bengal
5~ o 5 +6 7
0 S ~ 6 7 6 . 7 . c
Table 1~1 ~Example 13~
: ~ Log ( reduct ion ) . ` `.
Propan-2-ol ~ ~ Imbentin : ~ ~ ~After: No j.;.;.:
9 6 ~C9~t-35 :~:~ Light~ Ex:posure Rose::~Bengal
5 : ~ 0 ~ *6~3
i O: ~ O ~ S + 6 . 8
o.7 \ ~6.a r~
4 ~ 8
~ 5 ~ 5 0 ~ 3 jt. ``
1 0 ~ 3 ~ 0 1 ~ 9
0~ 2 1 ~3
0-5 1-0 ~1~2 ~
0 - 7 1 . 1 , 1 - 1 ;'~'
. 1'`,`
,~ ~ c
- W094/02022 PCT/GB93/01478 ~
21~08~G
- 33 - ~
::
Table 12 (Example 14) ~
. .,
. . .
Log (reduction) .
EthanolImbentin After No
: % C91-35 ~Light Exposure Rose Bengal
0.2 4.1 ~i.
0.6 +7.1 ,.
- 5.0 0.2 :~`
- +7.1 0.2
_ +7.1 2.2 -
- 0.2 3.3 2.5 .:~
- 0.6 3.4 2.6 :~
_ - 3 9
:: .
'.''~
.~
. ~
';'~
:
