PL173758B1 - Improvement in or in connection with compositions exhibiting killing properties in respect to micro-organisms - Google Patents

Abstract

A surface germicidal composition comprises a dyestuff which is capable of photo-dynamic inactivation of micro-organisms. The dyestuff preferably generates singlet oxygen on exposure to light and is substantive to microorganisms, conveniently being selected from the group comprising Rose Bengal, Erythrosin B and phthalocyanin sulphonates. The composition may optionally include other ingredients such as one or more surfactants (for cleaning purposes) and/or one or more solvents.

Description

The present invention relates to a method of destroying microorganisms deposited on a surface by inactivation.

The method of destroying microorganisms deposited on the surface according to the invention consists in:

a) treating the surface covered with the deposited microorganisms with a composition containing a dye selected from the group consisting of Rose Bengal, Erythrosine B and phthalocyanine sulphonates which binds microorganisms and causes photodynamic inactivation of microorganisms in an amount from 1 to 100 ppm; and next

b) the surface is exposed to light, killing microorganisms deposited on the surface by photodynamic inactivation.

Preferably, a dye is used which produces singlet oxygen when exposed to light, or a dye which is substitutable to microorganisms is used.

A dye that whitens by exposure to light is also preferred.

In a preferred embodiment of the process according to the invention, a composition is used which additionally comprises a surfactant, preferably an alkoxylated surfactant or an ethoxylated surfactant.

Another preferred surfactant is an agent that is at least predominantly non-ionic and / or anionic.

Preferably, the surfactant is used in an amount ranging from 0.05 to 2.5% by weight of the total weight of the composition.

In another preferred embodiment of the process according to the invention, a composition is used which comprises one or more solvents, preferably a polar solvent, or a straight chain or branched alcohol having 2 to 5 carbon atoms.

Preferably the solvent is used in an amount ranging from 2 to 20% by weight of the total weight of the composition.

In another preferred embodiment of the process according to the invention, a composition is used with a pH in the range from 3 to 5, preferably the pH is 4.

In a particularly preferred embodiment of the process, a dye composition is applied to the surface covered with deposited microorganisms, with a dye composition causing photodynamic inactivation of microorganisms and a surfactant and solvent, or an acidic, aqueous cleaning composition is applied to the surface covered with deposited microorganisms, which contains an acidic dye which is substitutable with respect to proteins, water-miscible solvent, and alkoxylated surfactant, and the surface is exposed to light and then rinsed.

It is especially advantageous to use a dye which produces singlet oxygen when exposed to light. By absorbing light energy, the dye molecule is converted, going from the basic electronic state (So) to the (Si) state. (Electron spins are paired, so in a spectroscopic image they appear as singlets having a single energy level in a magnetic field.)

The excited state is short lived and can, in many ways, lose energy by returning to the ground state, e.g. by emission of a light quantum during fluorescence, by internal conversion and energy loss during heating, by collision with a molecule of another substance (fluorescence quenching).

The unstable singlet state can also undergo a process called intercombination conversion to become a more permanent excited state, a triplet state.

173 758 (This state is referred to as a triplet because the spin of the higher energy level electron is not paired with the lower level electron spin, so the excited state has three energy levels in the magnetic field.)

The interaction of the excited triplet state with the ground state oxygen molecule (which normally occurs in the triplet) restores the dye to normal state after energy is transferred to oxygen, which is excited to the electronically excited singlet state. This means that under ideal conditions a single photosensitizer molecule can repeatedly generate its own singlet oxygen clusters.

A single oxygen atom is highly reactive and the process of photosensitized oxidation in this way is known as type II photooxidation. Type II photooxidation is independent of the photosensitizer used to generate singlet oxygen. An important feature of the photosensitizer is that it should have a high quantum triplet-forming efficiency (i.e. ideally every photon absorbed should produce a triplet). The presence of heavy atoms in the molecule enables intercombinational conversion to a triplet state.

Photoxidation of any vital components of the body can cause cell death (proteins, polypeptides, amino acids, lipids with allyl hydrogen atoms, tocopherols, sugars and cellulose).

Preferred dyes currently used include Rose Bengal (Acid Red 94, Color No. 45440), Erythrosine B, (Acid Red 51, Color No. 45430), and phthalocyanine sulfonates such as aluminum phthalocyanine sulfonates (APS) and zinc phthalocyanine sulfonates (ZPS). Rose Bengal and Erythrosine B are well-known food dyes (Rose Bengal is a food color labeled Food Red No.105 and Erythrosine B is Food Red No.14), with Erythrosine B being listed on the EEC list as a color approved for use in cosmetic products thus, both of these dyes are perfectly suitable for use in household compositions. A mixture of dyes may also be used, and in some cases it would be desirable to include a dye in the mixture which will remain visible until the photodynamic process is complete.

The concentration of the dyes in the composition is not critical and is usually up to 100 ppm, but good results have been obtained with concentrations in the range 10 ppm to 20 ppm. Lower concentrations, down to 1 ppm, can also produce satisfactory results.

Singlet oxygen is unstable and therefore has a short diffusion path, and therefore the photosensitive singlet oxygen-producing dye must be close to the basic substrate to be effective. Thus, preferred dyes are substantive to microorganisms (i.e. capable of binding) and usually bind to a cellular protein on the surface of the organism or else to other cellular components, e.g.

The preferred dyes mentioned above, when exposed to light, produce singlet oxygen and are protein substantive, and therefore capable of binding to microorganisms via the cellular protein. In this way, the deliberate destruction of the micro-organisms and thus the killing effect on the micro-organisms is possible.

It is also advantageous to use a dye that whitens on exposure to light. The use of a dye which bleaches under the influence of light and is substantive towards microorganisms allows obtaining a visible indicator of the presence of microorganisms. The photodynamic process runs along with the bleaching of the dye, causing the death or other inactivation of microorganisms. It has been found that in the case of lower light levels, both whitening and photodynamic effects are slower, while stronger light intensifies both of these processes, and they proceed much faster. Thus, depending on the relative bleaching rates and photodynamic activity, the presence of visible dye indicates to the user that photodynamic deactivation of any microorganisms present has not been completed.

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The photodynamic inactivation of microorganisms in suspension by means of a dye such as Rose Bengal is known. See, for example, Journal of Applied Bacteriology 1985, [58], pp. 391-400, Photochemistry and Photobiology 1988, [48] pp. 607-612 (Neckers et al.) And Shokuhin Eiseigaku Zasshi 1962, 10 (5), pp. 344-347. However, it has now been found that suitable dyes are capable of photodynamic inactivation of microorganisms on surfaces and this is the basis of the present invention. As is known, microorganisms are much more sensitive to killer agents when they remain in the plankton or suspended state: while they are much more difficult to inactivate when the surface to which they are attached is their natural or beneficial substrate. Usually, the microorganisms stay on the surface in the form of biofilms embedded in the matrix of the extracellular substance. This extracellular substance is sometimes referred to in the literature as adhesin. Thus, it is not obvious that a process that affects microorganisms in the plankton state without modification will be effective against surface attached organisms. Surface-borne microorganisms are an important and essential source of contamination in homes, institutions, and industrial environments, and the present invention may enable the intended antimicrobial activity to be achieved.

Methods for inactivating microorganisms by means of dyes are described, for example, in the publications EP-81 462, EP-286 524, DE-281 22 61. None of these publications presents the results of the effectiveness of the described compositions against microorganisms deposited on the surface, the activity tests carried out correspond to suspension tests used to test surface microorganisms in suspension.

In contrast to the known methods of inactivation of microorganisms by means of dyes, the method of killing microorganisms presented in this application with dyes selected from the group consisting of rose bengal, erythrosin B and phthalocyanine sulphonates relates to the destruction of microorganisms deposited on the surface.

During the research it was found that there are significant differences between the destruction of microorganisms deposited on the surface and the destruction of microorganisms present on the surface in the form of a suspension. Depending on the presence of microorganisms on the tested surface, different methods of biocide testing are used, i.e .:

suspension test - for testing agents on the organisms present on the tested surface in the form of a suspension; and the surface test - for testing microorganisms deposited on the tested surface. Examples of such assays have been described, inter alia, in the following publications: D. S. Dhaliwal, J. L. Cordier and L. J. Cox Impedimetric evaluation of the efficiency of disinfectants against biofilms in Letters in Applied Microbiology 1992, 15, 217-221; J. T. Holah, C. Higgs, S. Robinson, D. Worthington, and H. Spenceley 'A conductance-based surface disinfection test for food hygiene in Letters in Applied Microbiology 1990, 11, 255-259; or P. Maris Development of a micro-organism carrier-surface method in Science Des Aliments 1992, 12, 721-728.

The conducted research shows that microorganisms deposited on the surface are more difficult to remove, e.g. they cannot be removed by rinsing and rubbing the surface, and moreover, such microorganisms are more resistant to biocides (10 to 500 times more resistant depending on the type of tested biocide).

It has surprisingly been found that by applying the method according to the invention, by treating a surface with a composition containing a selected dye and exposing it to light, it is possible to photodynamically inactivate microorganisms deposited on the surface.

The method of the invention is particularly suitable for use in homes and industrial plants, on hard surfaces such as glass, plastic, ceramics and metal surfaces. He uses compositions particularly effectively

173 758 on surfaces that can harbor dirt, and they affect bacteriological contamination in kinks, junctions, and other relatively confined areas.

The compositions are preferably acidic and have a pH, e.g., in the range of 3 to 5, e.g. a pH of about 4, as acidic compositions have been found to have substantially enhanced efficacy against Gram-negative (G-) microorganisms when compared to neutral compositions. The efficacy against gram-positive (G +) microorganisms does not appear to be substantially pH dependent. Conveniently the compositions are acidified using a relatively mild organic acid such as acetic acid.

Neckers et al. (Supra) argue that the mere permeation of Rose Bengal pigment through the cell wall is essential for the inactivation process. As suggested, the results of their research confirm the hypothesis of dye penetration, mainly based on differences in inactivation of G + and G- species. The G- bacterial shell contains an additional diaphragm substantially composed of lipopolysaccharide which Neckers et al. Describes as a barrier to potential toxic substances. An alternative interpretation may be the fact that the amount of proteins with binding affinity for the dye present in the cell walls is very different for the G + and G- species, and higher for G + than for G-, and it changes with pH. The concentration of the dye bound to the cell wall would therefore be a function of pH. Such an interpretation would confirm our observations regarding the pH dependence of the kill rate (however not in the case of the particular G + resistance of B. subtilis and B. megaterium species).

There is no difference between the photodynamic sensitivity of Rose Bengal in endospore-forming B. subtilis species and non-spore-forming S. aureus when endospores are absent. The obvious differences shown in our research are presumably related to the presence of both B. subtilis and B. megaterium endospores, which are clearly more resistant to singlet oxygen than the bacteria themselves. Spores exposed to Rose Bengal and light stay alive and continue to germinate to form countable colonies. It is known that the spores are difficult to color and presumably, regardless of the conditions used, have no affinity for Rose Bengal. The toxicity of singlet oxygen to spores is not sufficiently documented in the literature. However, studies show that singlet oxygen in this respect has an effect on the much less resistant conidia of Neurospora crassa (Photochem. Photobiol., 33, 349, (1981)).

The composition may optionally contain other ingredients such as one or more surfactants (for cleaning) and / or one or more solvents.

The surfactants are preferably alkoxylated, more preferably ethoxylated, e.g. in the form of ethoxylated alcohols. The alcohol preferably has 4 to 15 carbon atoms, is in a straight or branched chain configuration and has an HLB (hydrophilic-lipophilic equivalent) value in the range 10 to 14, e.g. 12.

Many suitable surfactants are commercially available, one of which is that available under the tradename Imbentin 91-35 from Kolb, which is a C9.11 nonionic ethoxylated alcohol containing an average of 5 moles of ethylene oxide per mole of alcohol.

Primary ethoxy sulfates can also be used.

If desired, mixtures of surfactants can be used.

Preferably, the surfactant is non-ionic or anionic, or a mixture of both.

Preferred anionic surfactants include primary alkyl sulfates (PAS), preferably sodium dodecyl sulfate (SDS). Especially beneficial

173,758 are commercially available mixtures with significant dodecyl sulfate content (e.g. Empicol LX). Dodecyl sulfate is a known protein denaturing agent, it is effective at removing protein from surfaces and has a biocidal effect.

The composition is preferably substantially free of cationic surfactant, but may contain a small amount of cationic germicide.

The surfactant is preferably present in an amount in the range of 0.05 to 2.5% by weight of the total weight of the composition, usually from 0.5% to 1.5% by weight, e.g. 0.7% by weight of non-ionic, optionally up to 0.1% 2% by weight of anionic surfactant.

A preferred solvent is a polar solvent and is preferably a straight chain or branched alcohol containing 2 to 5 carbon atoms such as ethanol, butanol, isopropanol, (propan-2-ol) (IPA), N-butoxypropan-2-ol (ether n-butyl propylene glycol), 2-butoxyethanol (ethylene glycol monobutyl ether). The preferred solvent is IPA.

A dihydric alcohol such as ethylene glycol and water miscible ethers such as dimethoxyethane can also be used.

If desired, a mixture of solvents may be used, e.g. a mixture of ethanol and N-butoxypropan-2-ol.

Preferably, the amount of the solvent is in the range of 2 to 20% by weight based on the total weight of the composition.

At least some of the above solvents, e.g., ethanol, weaken the cell walls of microorganisms, making them more permeable and therefore more sensitive to singlet oxygen penetration. Thanks to this, the effect of destroying microorganisms by the dye is enhanced.

It has been found that the presence of a surfactant may reduce the photodynamic effect of the dye (possibly by dissolving the dye and thus preventing adsorption on the cell walls) and the presence of the solvent may also reduce such an interaction (presumably competing with singlet oxygen). However, it has been found that when a ternary composition is prepared containing a dye, a surfactant and a solvent, the reduction of the photodynamic effect of the dye is less than would be expected from the combined effect of the surfactant and solvent. Together, the surfactant and the solvent have a synergistic effect which results in a reduction in the reduction level of the photodynamic effect of the dye.

The composition may contain many additional components, namely:

1. Detergent boosters, preferably metal chelating agents such as ethylenediaminetetraacetic acid (EDTA). Metal chelators (including EDTA) have also been found to be able to penetrate cell walls, making microorganisms more sensitive to killer agents.

2. Electrolytes such as a buffer or salt, eg Na2SO4, which bind the dye to the protein, assisting its movement from the aqueous phase to the protein salt. The electrolyte is typically present in a variety of commercially available dye compositions, although additional electrolyte may be added if desired. The total electrolyte content of the composition will usually be in the range 0 to 1% by weight, preferably about 0.1%.

3. Fragrances

4. Thickening agents

The agent is in the form of an isotropic single-phase composition and is particularly used as a germicide (also with possible cleaning action) on hard surfaces, finding application in a broad context, including home use, e.g. kitchen and bathroom surfaces, including shells

173 758 toilets, in institutions such as schools, hospitals, etc., as well as in commercial premises such as factories, offices, hotels, etc.

For domestic use, the composition is preferably formulated as a sprayable product and is conveniently packaged in suitable containers, e.g., a spray can with a manually actuated latch or an aerosol dispensing device. Preferably the container is impermeable to light.

The composition is applied to the surface to be treated by any convenient means, e.g. by spraying from an appropriate dispenser, wiping with a carrier such as a cloth or sponge, or pouring from a container, etc. The surface is then exposed to a light source. for example white light sources such as a quartz halogen lamp or a fluorescent daylight source. (This method may be an alternative to the hazardous microbial radiation used, e.g. from a low pressure mercury lamp emitting resonance radiation at 254 nm. Such radiation is harmful to unprotected eyes). A washing step may then be applied if desired, e.g. by wiping with a carrier, or by applying a stream of running water, etc.

In order to illustrate the invention, it will now be described by means of the following examples and the related drawings, in which:

Figure 1 is two-bar plots of log (reduction) values illustrating the lethal effect of Rose Bengal and St. John's Baptism on various microorganisms in suspension at pH 4 and pH 7; Fig. 1a shows the results for Gram-positive organisms and Fig. 1b for Gram-negative organisms and yeast;

Figure 2 is a pair of log (reduction) plots showing the biocidal effect of rose bengal and light on 5. aureus and E. coli as a function of pH value; Fig. 2a shows the results after 20 minutes exposure to light and Fig. 2b shows the results after 60 minutes exposure;

Figure 3 is a pair of graphs as in Figure 2 obtained with the use of Erythrosin B in place of Rose Bengal;

Figure 4 - Log (reduction) two-bar graph illustrating the photohygienic interaction of various combinations of Rose Bengal (RB), Imbentin C91-35 (AE), isopropanol (IPA) and Empicol LX (PAS); Fig. 4a shows the results obtained in the absence of light, and Fig. 4b after exposure to light;

Figure 5 is a graph of the adsorption of Rose Bengal by E. coli, the adsorbed amount (nanomoles) of which is opposed to the equilibrated concentration (micromoles / 1), for the results at pH 4 represented by squares and at pH 7 marked with crosses;

Figure 6 is a pair of graphs, similar to Figure 5, showing the interaction of the electrolyte; Fig. 6a shows the results obtained at pH 4, and Fig. 6b at pH 7, with the results achieved without additive marked with squares, with sodium sulphate (1%) marked with crosses and sodium sulphate (5%) marked with double crosses;

Figure 7 is a pair of graphs, similar to Figure 5, showing the interaction of the surfactant; Figure 7a shows the results at pH 4, and Figure 7b shows the results at pH 7, with the results for the non-ionic surfactant (0.7%) (NI) marked with crosses and for PAS (0.7%) marked with double crosses ;

Figure 8 is a graph, similar to Figure 5, showing the interaction of the solvent at pH 4 with the results obtained without the use of additive marked with small squares, for 10% IPA marked with double crosses, for 0.7% Imbentin marked with double crosses and for 10% IPA and 0.7% Imbentin marked with larger squares;

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Figure 9 is a graph of the log (reduction) of exposure time (minutes) showing the effect of pH on the rate of destruction of E. coli by Rose Bengal, with the results at pH 4 marked with squares and at pH 7 marked with crosses, and

Figure 10 is a graph similar to Figure 9 showing the effect of electrolyte at pH 7 on the rate of destruction of E. coli by Rose Bengal, with electrolyte free results marked with squares and achieved with sodium sulphate marked with crosses.

In the description, examples and diagrams the following abbreviations are used:

- Benaal rose;

- Imbeniin C91-35;

- isopropanol;

- Empicol 1X5;

- there is a new surface agent

RB AE IPA PAS NI

The auxiliary agents used contain the following active substances:

Empicol LX Imbentin C91-35 Dowanol PnB Lialet 111 sodium lauric sulphate;

- E-oxyated head alcohol with D-H carbon atoms;

- N-butoxy propan-2-ol;

- e-alkoxylated alcohol with 11 otomas; gia and medium degree of ethoxylation 3.

Preparation of the inoculum

The following microorganisms [obtained from both the National Collection of Type Cultures (NCTC) and the American Type Culture Collection (ATCC)] were used in the experiments described:

Bacteria:

Staphylrcoccul aureus NTCT 6538 (Gram positive) Escherichia coli NCTC 8196 (Gram negative) Nickname aeruniaosa NTCT 5940 (Gram negative) Enterobacter sp. NTCT 3281 (Gram negative) Klebsiella sp. ATCC 11677 (Gram negative) Bacillus subtilis NCTC 6432 (Gram positive) Bacillus megaterium NCTC 7581 (Gram positive)

Yeast:

Cindida albicans

The organisms were grown overnight in broth at 37 ° C for bacteria (28 ° C for Ps. Aerugiaosa) or in SABS broth (SABS is Sabourand's dextrose agar, liquid medium for SABS broth from Oxoid Ltd) and temperature of 28 ° C for yeast. The cultures were separated by vacuum filtration using a 0.45 µm Millipore filter and washed with a quarter strength Ringer solution, then resuspended in Ringer's solution (10 mL). The organisms in the suspension were counted by serial dilution and plating into plates of nutrient agar (bacteria) and SABS agar (yeast), expressing the total number of viable organisms (TVC) as the logarithm of the number of colony forming units (cfu) per ml.

Unless otherwise stated, the experiments were conducted at a pH of 7.

1) Diffusion-disk agar method

Aqueous solutions containing 100 ppm of dye were prepared. An aliquot (10 ml) of each dye solution was sterilized in standard glass vials with a screw cap. Antibiogram discs (13 mm, produced by BDH) were also sterilized. All organisms were grown overnight in SABS medium or broth (10 ml).

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For each microorganism, the culture (10 µl) was inoculated overnight on two plates of nutrient agar, resulting in a flowing growth throughout the plate. The disc was immersed using aseptic techniques in the first dye solution and placed on the surface of the inoculated agar plate. The same was repeated with the other two dye solutions, making three discs on each of the duplicate plates.

One of each pair of duplicate plates was immediately placed in an incubator at the appropriate temperature and minimal exposure to light. The remaining plates were placed on top of the lit box for 3 hours. The illumination of the box was diffused white light with an average intensity of 4,000 lux from a daylight fluorescent lamp (2 x 15 watts, manufactured by Exal X-ray Accesories Ltd, Hemel Hempstead). The light intensities on the diffuser surface were measured using a Megatron DA10 light meter. Following exposure, plates were incubated overnight and then zones of inhibition around the disc were examined.

Using the above method, the following results were obtained:

Example 1. (Disk method)

Table 1 summarizes the agar results for the aqueous dye solutions: Rose Bengal, Erythrosine B and Aluminum Phthalocyanine Sulphonate (APS) (100 ppm) at pH and 180 minutes exposure to light as described above. The results in the table are expressed as the difference (in millimeters) between the radius of the pronounced inhibition zone (this is the area with no bacterial growth on the agar-coated plate) and the radius of the disc. The higher this value, the greater the kill of bacteria.

According to the agar-disk diffusion method, rose Bengal is positioned as more effective than the structurally similar erythrosine B. This may be attributed to a difference in the quantum efficiency of singlet oxygen production. The quantum yield of singlet oxygen production in methanol is 0.76 for rose bengal compared to 0.6 for erythrosine B. However, many other factors may also contribute to the observed discrepancies, such as dye diffusion rate or differences in dye-gel binding agar or disc material.

A special feature of the results of the disk method is the marked difference in the sensitivity of gram-positive and gram-negative organisms to photodynamic effects at a pH of at least 7. The putative reasons for the relative resistance of G- microorganisms are discussed elsewhere.

Test solutions for surface tests

Rose Bengal (20 ppm) in a pH 4 buffer and:

1) No other additives

2) Ethanol (10% v / v) and Imbentin C91-35 (0.7%)

3) Propan-2-ol (10% v / v) and Imbentin C91-35 (0.7%)

A sodium hypochlorite solution (0.125%) was used as a positive control solution.

Assessment of the number of organisms adhering to the surface of a Petri dish

A suspension of microorganisms, e.g. S. aureus (0.5 ml) was added to an aliquot (100 ml) of the 1/4 strength Ringer solution and the mean cfu per ml (TVC) was determined. Aliquots of these solutions (20 ml) were pipetted into sterile petri dishes and left for 5 hours at room temperature. The inoculum was then removed by pipette into a sterile bottle and the amount of cfu per ml remaining in suspension was calculated. Based on the difference in concentration of the solutions, the number of organisms adhering to the Petri dish (as cfu) per square cm was estimated.

Research method

The bacterial suspensions in 1/4 strength Ringer's solution (20 ml) were pipetted onto sterile plastic petri dishes and left at ambient temperature for 5 hours. Then the inoculum was removed, plates were washed once with Ringer's solution,

ΥΤ3 758 using gentle manual centrifugation, after which the solution was poured out. Pairs of bacterially contaminated plates were treated with the test solutions. An aliquot (20 ml) of the test solution was poured onto one plate and after 30 seconds the solution was decanted. The plate was washed with pH 4 buffer and placed in a lighted box for 20 min. In a separate experiment, the second plate with the solution was left for 20 minutes in the lighted box, and after decanting the solution, it was washed with a buffer at pH 4. Both experiments were repeated.

After exposure to light, one of the plates was covered with Tryptone Soya soy agar containing 1% glucose and 0.015% neutral red chilled to about 50 ° C. The second plate was stained for 30 seconds with 0.01% acridine orange, washed and examined under a microscope (Nikon Optiphot microscope equipped with a 100x apochromatic objective with immersion oil, a 10x eyepiece and an epifluorescence device with a double-sided filter B2-A / dichroic mirror system. and a high-pressure mercury lamp). The agar-coated plates were incubated at 37 ° C for 48 hours, at which time colonies grew from the non-destructed bacteria adhering to them. Colonies picked up neutral red and could be seen and counted under agar on the surface of the plate. (A. M. R. Mc Kenzie and R. L. Rivera-Calderon, Agar Overlay Method to Measure Adherence of Staphylococcus epidermidis to Four Plastic Surfaces, Applied and Environmental Microbiology, 50, 1322 (1985)).

Plates stained with acridine orange were examined and photographed. The colored bacteria in the field of view (clusters treated as one) were counted after being assessed by photographing on a micrometer scale.

Example 2 (Surface Test)

Control surface studies, both using direct epifluorescence microscopy as described above and drying the slurry, gave similar results for the amount of bacteria (S. aureus) attached to the surface of the plastic plate (on the order of 1 million per square cm).

Reducing the surface area of the positive control (sodium hypochlorite solution, 30 seconds) reduces the number of viable bacteria to zero. The results for the photodynamically inactivated bacteria are summarized in Table 2 as the decimal log reduction [log (reduction)] of viable bacteria before and after exposure, i.e. the log (starting amount) log (final amount).

Example 3. Composition: Rose Bengal (20ppm), non-ionic surfactant (Imbentin C91-35, 0.7%), propan-2-ol (10%) (pH 4)

The experimental procedure described previously was used, except that the bacteria needed to be in a vigorous growth phase to be attached to the surface. This goal was achieved by incubation for 3 hours in plastic petri dishes in broth (for bacteria) or SABS broth (for yeast). In Example 2, the suspension of non-growing bacteria in Ringer's solution was allowed to stand for 5 hours. This worked well for S. aureus, but not for other bacteria. The results are presented in Table 3.

Previous experience with direct epifluorescence microscopy suggests that, if diffuse growth is obtained, it could be equivalent to an initial viable surface density of 1 million / cm ² . The area of the Petri dishes used was about 57 cm ² , so that in all cases the treatment reduced the level of contamination and the order of magnitude was at least 5 (log 5).

Surface studies are much more difficult to perform than slurry studies, therefore, to show the effect of changing conditions, most of the experimental work was conducted in slurry.

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Preparation of solutions for testing in suspension

The following stock solutions were prepared by weighing and sterilizing (except those containing solvent):

Rose Bengal (0.2%) in propane-2-ol (95%)

Non-ionic surfactant (Imbentin C91-36, 14%) (sometimes abbreviated to AE for ethoxylated alcohol)

Anionic surfactant (Empicon LX, 14% (sometimes referred to as PAS) buffer pH 4 [citric acid (0.1 M, 307 ml) + dibasic sodium phosphate (0.2 M, 193 ml) buffer pH 7 [sodium dihydrogen phosphate (0.4 M, 468 ml) + disodium hydrogenorthophosphate dodecahydrate (0.4 M, 732 ml)]

Buffers at 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).

The final concentrations of the test solutions were usually:

Rose Bengal 20 ppm

Ethanol 10.0% (µm / ti)

Surfactant 0.7% (w / v)

In several examples, concentrations other than the specified ones were used.

Research method

The solutions were prepared in 5 mm deep sterile plastic petri dishes (30 bowls). The microorganism suspension (0.3 ml) was added to each solution and mixed gently. If Rose Bengal was present in the test solution, it was added last to minimize exposure. Then the solutions were either placed in a lighted box, in the dark (under reduced light exposure conditions), or left on a table. The average light intensity on the surface of the diffuser in the illuminated box was 4000 lux, measured with a Megatron DA 10 light meter (manufactured by Megatron Ltd). After the specified exposure times, viable bacteria were counted as colony forming units (cfu / ml), then incubated, serially diluted and plated on agar plates. The decimal logarithm of the amount of bacteria remaining (as colony forming units per ml) was determined and compared with the amount before exposure - log (starting amount) - log (final amount). The higher this value, the greater the kill of bacteria.

To optimize the photodynamic activity against a wide variety of organisms, including gram-negative organisms and yeasts, suspension studies have been conducted with a variety of microorganisms and under a variety of conditions. The results of these tests are summarized in the appropriate tables. They are expressed as the logarithm of the initial number of colony forming units per ml to the amount remaining after exposure, log (reduction). According to this labeling, a value of zero means that the number of organisms has not changed after being exposed to different conditions. A label followed by the number of log reduction indicates that no growth of the microorganisms (i.e. complete kill) was observed.

Example 4. In order to demonstrate the lethal effect of Rose Bengal and light on the microorganisms in the suspension, and especially the synergy of low pH and ethanol, a suspension study was performed as described above. The studies were conducted using rose bengal alone (20 ppm) and in combination with ethanol (10%, v / v) and with a 20-minute exposure to light (illuminated box). The results expressed as log values (reduction) are given in Table 4.

Longer exposure times (60 and 100 minutes) improve the results for Gram-negative organisms, especially at pH 7.

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As will be shown, the results for Gram negative organisms are significantly better at pH 4 compared to pH 7.

Example 5 The tests were conducted as described above at pH 4 and pH 7 and the lethal effects of Rose Bengal (20 ppm) and light (20 minutes exposure in an illuminated box) against a variety of Gram-positive and Gram-negative microorganisms and yeasts in suspension. The results expressed as log (reduction) are shown graphically in Figures 1a and 1b. Fig. 1a shows the results for gram-positive microorganisms and Fig. 1b for gram-negative microorganisms and yeast.

Example 6 Tests were carried out as described above at various pH values and the biocidal activity of rose bengal (20 ppm) and light on

S. aureus (G +) and E. coli (G-) as a function of pH. The results, expressed as log (reduction), are shown graphically in Figs. 2a (20-minute exposure) and 2b (60-minute exposure). In these figures, the crosses show the results for the controls (G-), the double crosses for the E. coli, the inverted triangles for the controls (G +), and the triangles for

S. aureus ;.

Similarly, suspension studies were performed to demonstrate the biocidal activity of erythrosin B and light on S. aureus and E. coli as a function of pH. The results are shown graphically in Figs. 3a and 3b, which are almost identical to Figs. 2a and 2b. They show that erythrosin B acts in a similar way to rose bengal in terms of its photobiocidal pH profile.

Example 7 Further slurry tests were conducted as described above at pH 7 and using the following solutions:

Imbentin C91-35 non-ionic surfactant (0.7%)

Imbentin C91-35 (0.7%) with Rose Bengal (100 ppm)

Empicol LX anionic surfactant (0.7%)

Empicol LX with Rose Bengal (100 ppm)

Imbentin C91-35, Empicol LX (both 0.7%) and Rose Bengal (100 ppm)

The results are summarized in Table 5 below. In this table, the term dark indicates the conditions of reduced exposure to light rather than total darkness, due to the practical difficulties in avoiding even slight exposure to light. The term clearly means the results that are the average of several experiments performed over the time range (20 minutes, 1 hour, 3 hours) in a statistically determined experiment.

Results obtained in a similar manner for E. coli at pH 7 are graphically represented as bar graphs in Figure 4. In this figure, PAS is used as an abbreviation for Empicol LX, IPA is an abbreviation for isopropanol, and AE for Imbentin C91-35. This figure shows the averaged data for 0.7% AE, 0.7% PAS, and 10% IPA.

Example 8 Further slurry studies were conducted at pH 4 for E. coli using one or more of the following: Rose Bengal 40ppm, 0.7% surfactant (Imbentin C91-35 or Empicol LX), IPA (isopropanol) 10 %. The results are given in Table 6.

The following examples are for suspension studies conducted generally as described above, but at a pH of 4. When Rose Bengal was used, it was present at a concentration of 20 ppm, although in some cases the control solutions were exposed to light, without the Rose Bengal, as shown in the column headed Without Bengal Pink.

Examples 9, 10, 11 and 12 used gram-positive S. aureus and examples 13 and 14 used gram-negative E. coli. Other reagents used are specified in the examples. In all these examples, the samples were exposed for 20 minutes in a lighted box. The average intensity on the diffuser surface was 4000 lux, measured with a Megatron DA10 light meter (manufactured by Megatron Ltd.).

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Example 9. Suspension studies were conducted for S. aureus using Rose Bengal, Ethanol and Imbentin C91-35. The decimal logarithm of the initial concentration, log (start), for S. aureus was 6.8. The results are given in Table 7.

Example 10. Suspension studies were conducted for S. aureus using Rose Bengal, Dowanol PnB and Imbentin C91-35. The log (start) was 6.9. The results are given in Table 8.

This example shows that Dowanol PnB has certain biocidal properties.

Example 11. Suspension studies were conducted for S. aureus using Rose Bengal, Ethylene Glycol and Imbentin C91-35. The log (start) was 6.8. The results are given in Table 9.

Example 12. A suspension study was conducted with S. aureus using Rose Bengal, IPA and Lialet 111. Lialet 111 is the name of a commercially available Enichem ether sulfate composition having an average chain length of 11 and an average degree of ethoxylation of 3. The log (start) was 6 , 7. The results are given in Table 10.

Example 13. Suspension studies were conducted with E. coli using Rose Bengal, Propan-2-ol and Imbentin C91-35. Log (start) is; 6.8. The results are given in Table 11.

Example 14. Suspension studies were conducted for £. coli using Rose Bengal, Ethanol and Imbentin C91-35. The log (start) was 7.1.

The results are given in Table 12.

Example 15. The adsorption of rose bengal was determined by evaporating water from a concentrated solution. Concentrations were obtained by spectroscopy from the absorbances measured at the maximum absorbance wavelength (about 549 nm) using a WPA Linton 3100 spectrophotometer, on supernatants freed from microorganisms by centrifugation.

The results are shown in Figures 5 to 8.

These results show that the photobiocide effect is dependent on the adsorption of the dye. Dye adsorption is:

a) increased at low pH (Figure 4);

b) enhanced by a neutral electrolyte (which also increases the photobiocidal effect on E. coli at neutral pH) (Figure 6);

c) decreased by surfactant (Fig. 7);

d) enhanced with propan-2-ol (Fig. 8).

The calculations indicate that the amount adsorbed by E. coli is in the correct proportion to protect the bacterial layer, and since the dye is known to have a tendency to accumulate, the calculated E coli surface area should be reduced (not taking into account the fimbriae / pili). A detailed breakdown of the results is given below. The molecular size of the Rose Bengal was calculated from a scale model (Catalin Ltd., London).

E. coli surface 1E7 nm ²

Bengal pink surface 2 im? (flat)

0.5 nm ² (side)

5E6 (flat) or 20E6 (side) protected layer

Measurements of adsorption 2-8 E-16 moles / bacteria

Number of particles 1210 - 480 E6 for bacteria

Example 16 A slurry study was performed as described above to determine the effect of rose bengal on the kill rate of E. coli as a function of pH and electrolyte addition (5% sodium sulfate) at pH 7 and the results are shown graphically in Figures 9 and 10, respectively.

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Table 1 (Example 1)

Photobiocidal impact of the disclosed agents

Inhibition zone around the disc

Microorganism Type Gram Pink Bengali Erythrosine B APS S. aureus + 4 1 2 B. subtilis + 3 1 1 B. megaterium + 3 1 1.5 E. ooli - 0 0 0 Ps. aeruginosa - 0 0 0 Enterobacter sp. - 0 0 0 C. albicans 0 0 0

Table 2 (Example 2)

Lethal effects of rose bengal and light on Staphylococcus Aureus attached to a plastic surface

Solution Log (Ratio) Rose bengal 4.7 Rose Bengal + Imbetin C91-35 + Ethanol 6.0

Table 3 (Example 3)

Microorganism No experience Results in agar-coated plate methods Control After the exposure S. aureus 1 spreading growth no growth 2 spreading growth no growth E. coli 1 Big increase 45 cfu 2 spreading growth no growth K. pneumoniae 1 spreading growth no growth 2 spreading growth no growth P. aeruginosa 1 Big increase 5 cfu 2 spreading growth no growth C. albicans 1 spreading growth 5 cfu 2 spreading growth 4 cfu

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Table 4 (Example 4)

Mikeoorganism Type Gram Exposure conditions No solvent With ethanol pH 7 pH 4 pH 7 pH 4 S. aureus + 7.0 7.1 7.1 6.9 B. subtilis + 0.6 2.1 3.1 0.8 B. megaterium + 1.3 0.3 1.1 0.3 E. coli - 0.3 5.3 0.1 6.9 K. pneumoniae - 0.9 5.6 0 7.0 Ps. aeruginosa - 0 7.0 0 6.9 Enterobacter sp. - 1.1 7.3 0 7.4 S. albicans - 0 5.7 0 5.7 Control (no pink bengal) E. coli 0.4 0.8

Table 5 (Example 7)

Microorganism IMBENTIN IMBENTTN / RB EMPICOL EMPICOL / RB brightly dark brightly dark brightly dark brightly 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 Klebsiella 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 0 0 0 0 0 0

Microorganism IMBENTIN / EMPICOL IMBENTIN / EMPICOL / RB PINK BENGALI brightly dark brightly dark brightly dark St. aureus 3.0 4.0 2.0 0 4.0 0 E. coli 4.0 4.0 4.0 4.5 0 0 Entero. 0 0 0 0 0 0 Klebsiella 3.5 3.5 4.0 2.5 0 0 Ps. aerug. 0 0 0 0 0 0 C. albicans 0 0 0 0 0 0

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Table 6 (Example 8)

EXPOSURE TIME 1 HOUR 2 HOURS THREE HOURS brightly dark brightly dark brightly dark Rose bengal 3.8 0.5 7.2 0.4 0.4 7.2 RB / Imbentin 0.2 0.2 1.0 0.3 3.8 0.5 C91-31 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 0.1 0.3 0.9 C91-35 Empicol LX 0.1 0.6 1.0 IPA 0.3 1.7 4.4

Table 7 (Example 9)

Ethanol% Imbentin C91-35% Log (reduction) After exposure to light Without pink bengal 5 0.2 +6.8 5 0.6 4.6 10 0.6 +6.8 15 0.6 +6.8 5 - +6.8 0.1 10 - +6.8 -0.4 15 - +6.8 0.0 - 0.2 4.6 2.5 - 0.6 3.5 2.3 - - +6.8 2.3

Table 8 (Example 10)

Dowanol% Imbentin C91-35% Log (reduction) After exposure to light Without pink bengal 3 0.7 +6.9 • 3 - +6.9 4.0 - 0.7 5.2 3.4 - - +6.9

Table 9 (Example 11)

Ethylene glycol % Imbentin C91-35% Log (reduction) After exposure to light No pink bengal 10 0.7 +6.8 10 - +6.9 -0.2 - 0.7 +6.8 3.4 - - +6.8

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Table 10 (Example 12)

Propan-2-ol % Lialet 111 % Log (reduction) After exposure to light Without pink bengal 15 0.5 +6.7 15 - +6.7 4.9 - 0.5 +6.7 +6.7 - - +6.7

Table 11 (Example 13)

Propan-2-ol % Imbentin C91-35 % Log (reduction) After exposure to light Without pink bengal 5 0.1 2.3 10 0.1 +6.8 10 0.5 +6.8 10 0.7 +6.8 - - 4.8 5 - 5.5 0.3 10 - 3.0 1.9 - 0.1 1.2 1.3 - 0.5 1.0 1.2 - 0.7 1.1 1.1

Table 12 (Example 14)

Ethanol% Imbentin C91-35% Log (reduction) After exposure to light Without pink bengal 5 0.2 4.1 15 0.6 +7.1 5 - 5.0 0.2 10 - +7.1 0.2 15 - +7.1 2.2 - 0.2 3.3 2.5 - 0.6 3.4 2.6 - 3.9

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^ KONTROWTG-] ^ E.coliVKONTROWG +) AS.aureus

Fig. 2b

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Fig. 4a

Fig. 4b

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Fig. 5

-o-NO ADDITIONAL | _ SODIUM SULPHATE (1%) ^ 'SODIUM SULPHATE ^ o / ^

Fig. 6a

Fig. 6b

- «SRAK ADDITION- + SODIUM SULPHATE H%) - SODIUM IARATE 5%)

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-o-NO ADD -HNI (0.7 ° / o) - ^ PAS (0.7%) I.

KU j

Fig. 7a

Fig. 7b

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-ο-srak tax- 1Q% IPA 0.7% Imbentin -ą-10% IPA / 0.7% Imbentin

Fig. 8

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* B§ $ TKU + SULPHATE Na ( ₅ ο _{/ ο)}

Fig. 10

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LOG / reduction /

Fig. 1a

Fig. 1 b

Publishing Department of the UP RP. Circulation of 90 copies

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Claims (17)

Patent claims 1. Method for destroying microorganisms deposited on the surface, characterized in that: a) treating the surface covered with the deposited microorganisms with a composition containing a dye selected from the group consisting of Rose Bengal, Erythrosine B and phthalocyanine sulphonates which binds microorganisms and causes photodynamic inactivation of microorganisms in an amount from 1 to 100 ppm; and next b) the surface is exposed to light, killing microorganisms deposited on the surface by photodynamic inactivation. 2. The method according to p. The process of claim 1, wherein the dye is used which generates singlet oxygen when exposed to light. 3. The method according to p. A method as claimed in claim 1 or 2, characterized in that the dye is substitutable to microorganisms. 4. The method according to p. The process of claim 1, wherein the dye is bleached by exposure to light. 5. The method according to p. The process of claim 1, wherein the composition is used which additionally comprises a surfactant. 6. The method according to p. The process of claim 5, wherein the alkoxylated surfactant is used. 7. The method according to p. The process of claim 6, wherein an ethoxylated surfactant is used. 8. The method according to p. A surfactant according to claim 5, 6 or 7, characterized in that the surfactant is at least predominantly non-ionic and / or anionic. 9. The method according to p. 5. The method of claim 5, 6 or 7, wherein the surfactant is used in an amount ranging from 0.05 to 2.5% by weight of the total weight of the composition. 10. The method according to p. The process of claim 1, wherein the composition is comprised of one or more solvents. 11. The method according to p. The process of claim 10, wherein the polar solvent is used. 12. The method according to p. The process of claim 11, wherein the solvent is a straight or branched chain alcohol having 2 to 5 carbon atoms. 13. The method according to p. 10, 11 or 12, characterized in that the solvent is used in an amount ranging from 2 to 20% by weight of the total weight of the composition. 14. The method according to p. The process of claim 1, wherein the composition has a pH in the range of 3 to 5. 15. The method according to p. The process of claim 14, wherein the composition has a pH value of 4. 16. The method according to p. The method of claim 1, characterized in that the dye composition causing photodynamic inactivation of the microorganisms and the surfactant and solvent is applied to the surface covered with the deposited microorganisms. 17. The method according to p. The method of claim 1, characterized in that on the surface covered with the deposited microorganisms 173 758, a) an acidic, aqueous cleaning composition is applied that contains an acidic dye that is substitutable with the protein, a water-miscible solvent, and an alkoxylated surfactant b) the surface is exposed to light and then rinsed.