A composition for biofilm disruption

Abstract

The present invention relates to a composition that is used to disrupt biofilm especially mixed biofilm that tends to accumulate on surfaces in the home like those in the bathroom, toilet, and kitchen sinks. This is achieved through a composition that comprises specific bacteriophage, a polysaccharide selected from one or both of pectin and resistant dextrin and a surfactant.

Description

[0001] A COMPOSITION FOR BIOFILM DISRUPTION

[0002] Field of the Invention

[0003] The present invention relates to a composition that is used to disrupt biofilm. Particularly, the present invention relates to a composition that disrupts mixed biofilm that tends to accumulate on surfaces in the home like those in the bathroom, toilet, and kitchen sink.

[0004] Background of the Invention

[0005] People like the surfaces of homes to be kept clean and free of dust and soil. Additionally, they also like to have cleanliness in other common spaces like offices, restaurants, hotels and public places like bus and train stations and airports. Firstly, consumers like to have a good visual appearance of cleanliness and apparent hygienic conditions in addition to the place smelling fresh and pleasant. Further, there are places in the bathroom, toilet and kitchen sinks which are generally wet most of the time where one encounters the formation of biofilm. Biofilm formation is a phenomenon where tough stains are formed as a consequence of proteins or carbohydrates that stick on to the surface often including therein bacteria that help the biofilm adhere very strongly on to such surfaces. Mixed biofilms are the ones formed where one encounters a combination of microbes like bacteria and fungi. These types of mixed biofilms are even more difficult to disrupt as compared to biofilms formed from only one type of microbe. In addition to microbiological methods of disruption, these biofilms often require aggressive mechanical scrubbing to remove them.

[0006] Conventional methods of cleaning like using surfactants, bleaching agents like chlorine or peroxides are not very effective in disrupting such types of mixed biofilms. Some antibacterial agents could be used but they often act only on one type of microbe e.g. a bacteria but the fungi is left unaffected thereby retaining the tough integrity of the biofilm. It is thus a challenge to disrupt mixed biofilms.

[0007] The present inventors in seeking to solve the above problem found to their surprise that use of bacteriophages that specifically lyse the bacteria present in the biofilm in combination with certain polysaccharides in a surfactant containing composition is able to disrupt mixed biofilms. Bacteriophages are bacterial viruses that attach only to their specific host bacteria and kill them by bacterial lysis. Phages are thus very specific in that they only attack their targeted bacterial hosts. They cannot infect humans or other eukaryotic cells. While it may be considered obvious to use such bacteriophages to disrupt bacterial biofilms, the surprising benefit of the present invention was its ability to disrupt mixed biofilm containing bacteria and fungi. Further benefit is observed in that the disruption is seen to occur to a high degree in much shorter time frames e.g. in about 8 hours as compared to individual ingredients taking about 24 hours to achieve even a small amount of disruption.

[0008] Thus, the use of such natural and ecofriendly actives provides for control of undesirable mixed biofilm. The present approach of using bacteriophages and polysaccharides thus offers a good sanitation method that is sustainable, effective in removing biofilm containing bacteria and fungi while offering a solution which has minimal side effects usually caused by usage of chemical actives.

[0009] It is therefore an object of the present invention to provide for a composition for disrupting mixed biofilm. of the Invention

[0010] The first aspect of the present invention relates to a liquid composition for disruption of mixed biofilm comprising bacteria and fungus comprising

[0011] (i) Bacteriophage capable of lysing bacteria selected from one or more of M. morganii, C. koseri, P. mirabilis, P. vulgaris and E. coir,

[0012] (ii) polysaccharide selected from one or both of pectin and resistant dextrin;

[0013] (iii) 0.1 to 25 wt% surfactant; and

[0014] (iv) 60 to 99% water; wherein the composition has a pH in the range of 4.0 to 10.0.

[0015] Another aspect of the present invention relates to the method of disrupting biofilm comprising the step of contacting the surface with a composition of the first aspect preferably diluted with water.

[0016] Detailed Description of the Invention

[0017] For the avoidance of doubt, any feature of one aspect of the present invention may be utilized in any other aspect of the invention. The word "comprising" is intended to mean "including" but not necessarily "consisting of” or "composed of'. Thus, the term "comprising" is meant not to be limiting to any subsequently stated elements, but rather to optionally also encompass nonspecified elements of major or minor functional importance. In other words, the listed steps or options need not be exhaustive. Whenever the words "including" or "having" are used, these terms are meant to be equivalent to "comprising" as defined above. It is noted that the examples given in the description below are intended to clarify the invention and are not intended to limit the invention to those examples per se. Except in the examples, or where otherwise explicitly indicated, all numbers in this description indicating amounts of material or conditions of reaction, physical properties of materials and / or use are to be understood as modified by the word "about". Unless specified otherwise, numerical ranges expressed in the format "x to y" are understood to include x and y. When for a specific feature multiple preferred ranges are described in the format "x to y", it is understood that all ranges combining the different endpoints are also contemplated. Unless specified otherwise, amounts as used herein are expressed in percentage by weight based on total weight of the composition and is abbreviated as “wt%”.

[0018] The composition as per the present invention comprises a bacteriophage capable of lysing bacteria selected from one or more of M. morganii, C. koseri, P. mirabilis, P. vulgaris and E. coli. Preferably, the composition comprises bacteriophage capable of lysing bacteria C. koseri and a bacteriophage capable of lysing bacteria E.Coli. More preferably, the composition comprises bacteriophage capable of lysing bacteria C. koseri and a bacteriophage capable of lysing bacteria P. vulgaris and a bacteriophage capable of lysing bacteria E.Coli. Most preferably the composition comprises a mixture of bacteriophages capable of lysing each of the bacteria M. morganii, C. koseri, P. mirabilis, P. vulgaris and E. coli. The bacteriophage is preferably included in an amount in the range of 102to 1011PFU / ml of the composition.

[0019] Biofilms are microbial aggregates which include heterogeneous populations of bacteria, fungi, and algae. They are ubiquitous in nature and represent populations of cells that have functional interdependencies upon one another which collectively offer microbial activities that are not possible by any of the individual component species. The biofilm matrix surrounding bacteria and fungi makes them tolerant to harsh conditions and resistant to antimicrobial treatments. The formation of microbial biofilm presents a challenge to the establishment and maintenance of hygienic conditions in public health, the home and in industry. In indoor environments, biofilms are formed on surfaces which include kitchen surfaces, drainages and sinks, dishwash sponges, refrigerators, washing machines, toilets, damp surfaces and ceilings and poses a threat to human health. Biofilms can thus form on hard as well as on soft surfaces. The occurrence of many biofilm-based infections and their multiple antimicrobial resistance is a major concern to human health. It is critically important to design or screen anti-biofilm molecules or compositions that can effectively minimize and eradicate biofilm related infections. Sometimes very aggressive mechanical scrubbing may be required in addition to harsh chemicals to remove such tough biofilms. It is observed that it is very difficult to disrupt biofilms and kill the germs present therein if one wishes to use agents that are mild on skin and safe for use. While it is known that bacteriophages are very specific in that they are capable of lysing only one type of bacteria, the use of such bacteriophages for disrupting mixed biofilm (which contain both bacteria and fungi) has not been known heretofore. Therefore, it came as a surprise to the present inventors that a combination of a specific type of bacteriophage and a specific polysaccharide as claimed herein in a cleaning composition was found to disrupt mixed biofilm.

[0020] The composition of the invention preferably comprises a polysaccharide which is selected from one or both of a pectin and resistant dextrin.

[0021] Pectin is a common polysaccharide found in many plants in nature. Various sources of pectin are available, including apples, citrus peel, gooseberries, quince, pears and plums. Pectin for use in the present invention may be from any of the above sources, preferably from citrus peel, apple or sugar beet, or mixtures thereof. Pectin consists predominantly of a-D galacturonic acid units, but also contains some amount of neutral sugars such as rhamnose, xylose, arabinose, galactose and glucose.

[0022] The percentage of galacturonic acid units is therefore the amount of galacturonic acid groups present relative to the total amount of the pectin. The galacturonic acid content of a sample can be determined by methods known in the art, such as for example the Saeman hydrolysis method (Englyst and Cummings (Analyst, 109(7), 937-942 (1984), Filisetti-Cozzi and Carpita (Analytical Biochemistry, 197, 157-162 (1991)).

[0023] Galacturonic acid typically has the following repeat unit structure: It is possible to esterify galacturonic acids in the carboxylic acid group. The percentage of esterified units is called the degree of esterification (DE). The degree of esterification can be determined according to methods known in the art, such as the base titration method (Shultz, 1965) as proposed by the Food Chemical Codex (FCC (1981). 3rd ed., (1981) National Academy of Science, Washington, DC), quantification of methanol released during de-esterification using gas chromatography (GC) (Walter et al. (1983), Journal of Food Science, 48: 1006-10070), colorimetry (Hou et al. (1999), Botanical Bulletin of Academia Sincia, 40:115-119), high performance liquid chromatography (HPLC) (Levigne S., et al. (2002), Food Hydrocolloids 16: 547-550), nuclear magnetic resonance (NMR) (Rosenbohm et al. (2003) Carbohydrate Research, 338: 637-649) and capillary zone electrophoresis (CZE) (Williams et al. (2003), Journal of Agricultural Food and Chemistry, 51 : 1777-1781).

[0024] A method to separate pectin into fractions with different DE is described, for example, by Strom, et al. (2005), Carbohydrate Polymers, Volume 60, Issue 4, 20 June 2005, Pages 467-473.

[0025] In nature, the carboxylic groups are typically methylated to varying degrees to provide pectin methyl esters. The number fraction of carboxylic acid groups methylated is known as the degree of methyl esterification.

[0026] The pectin used in the composition of the present invention has a degree of methyl esterification higher than 50 wt%, more preferably 52.5 wt% or higher, furthermore preferably 55 wt% or higher, yet more preferably 57.5 wt% or higher and yet furthermore preferably 60 wt% or higher, and most preferably 62.5wt% or higher.

[0027] The pectin used according to the invention has a molecular weight of higher than 50kDa, preferably higher than 100kDa, more preferably higher than 150 kDa. Typical upper limits of pectin molecular weights are of the order of 1000kDa, preferably 800kDa. Thus, the pectin for use in the composition of the invention has a molecular weight of between 50 kDa and 1000kDa.

[0028] The molecular weight of the pectin used in the present invention is determined using Triple Detection Size Exclusion Chromatography (TD-SEC)

[0029] The analysis is conducted using the Malvern OMNISEC system. The complete OMNISEC system consists of OMNISEC RESOLVE (chromatography module) and OMNISEC REVEAL (detectors module). Using multi-detector technologies, it combines refractive index, absorbance, light scattering and viscometer detectors to measure concentration, molecular weight and other parameters. The data are analyzed using the OMNISEC software.

[0030] Depending on the molecular weight of the polysaccharide, appropriate aqueous columns such as the A6000 and A7000 set of columns and the corresponding guard columns are used. Depending on the nature of the polysaccharide to be analyzed (whether they have anionic, neutral or cationic functional groups), routinely used eluents of choice are demineralized water containing 0.02wt% Na azide as the preservative or buffer solutions such as Na or Li nitrate (0.05-0.1 M), buffered to the appropriate pH range using NaOH or acetic acid. Polysaccharides with hydrophobic functionality can be analyzed using 10-20% MeOH.

[0031] Depending on the molecular weight range of the polysaccharides, typical sample solutions are prepared at 0.5 mg / ml and then filtered using 0.45pm Nylon filters prior to dispensing into sample vials. The eluent is filtered using a 0.2 pm Nylon filter. The flow rate throughout all runs is typically at 1ml / min; the detector and column oven temperatures are set to 40°C.

[0032] The detector calibration is carried out using a narrow molecular weight standard to calculate the detector offsets, the detector constants for all detectors and the band broadening and tailing corrections. The calibration is performed using a standard of known concentration, molecular weight, dispersity and intrinsic viscosity; in this case the narrow molecular weight standard of choice usually is a Pullulan standard material.

[0033] A verification standard is also run with every sequence. This standard is usually a broad (polydisperse) standard, whose properties are known and can be measured. This independently verifies that the calculation method has been correctly calibrated. For polysaccharide analyses, a Dextran 73KDa material is chosen.

[0034] For accuracy (and also to allow for the possibility of sample impurities being present), a fixed dn / dc method is used to calculate the molecular weight data for all samples assuming a dn / dc of 0.147.

[0035] The polysaccharide may also be resistant dextrin. Resistant dextrin is also known by some as digestion resistant maltodextrin. It is usually prepared by highly controlled partial hydrolysis of corn, potatoes, barley or wheat preferably from corn starch. It usually contains 3 to 20 D-glucose units that are linked by a(1 -4), a(1 -6), p(1 -4) and p(1 -6) glycosidic bond. It is also reported to contain some 1 ,2 and 1 ,3 glycosidic bonds. It usually has the chemical formula C6nH( n+2)O(5n+i) or (C6HioOs)n where ‘n’ represents the number of glucose units linked together. . It is usually sold as an off-white powder which is odourless. It is soluble in water. A preferred supplier of resistant dextrin is ADM Bio Science & Technology (Tianjin) Co. Ltd., Tianjin, China who sell it under the brand name Fibersol or Fibersol-2.

[0036] Of the polysaccharides, pectin is preferred for inclusion in the composition of the invention. Pectin when included is preferably derived from one or more of a fruit chosen from pear, apple, guava, plum, gooseberry, orange or any other citrus fruit, preferably citrus. It is preferably from apple or citrus fruit. The composition preferably comprises polysaccharide which is a combination of pectin and resistant dextrin. The polysaccharide is preferably included at a concentration of 0.01 to 20%, preferably 0.1 to 10% by weight of the composition.

[0037] The composition of the invention preferably comprises a surfactant. The surfactant that is used depends on the type of surface being cleaned. The surfactant may be one or more of anionic, non-ionic, amphoteric or zwitterionic type. It has been observed that the most preferred surfactant for use in the present invention, independent of the type of surface or type of soil to be cleaned, is an anionic or non-ionic surfactant, preferably a non-ionic surfactant. A non-ionic surfactant of the alcohol ethoxylate is especially preferred. Preferably, the composition comprises from 1 to 10 wt% non-ionic surfactant based on the total weight of composition. A preferred class of nonionic surfactant for use in the invention includes aliphatic Cs to Cis, more preferably C12 to C15 primary linear alcohol ethoxylates with an average of from 3 to 20, more preferably from 5 to 10 moles of ethylene oxide per mole of alcohol. The formulation of the composition with a non-ionic surfactant is found to deliver superior biofilm disruption as compared to an anionic surfactant.

[0038] The composition of the invention may preferably comprise an organic acid or its salts. Preferably, the organic acid or its salt is selected from one or more of citric acid, succinic acid, malic acid, lactic acid, tartaric acid, hexanoic acid, cyclohexanoic acid, heptanoic acid, octanoic acid, 4- methyl octanoic acid, nonanoic acid, decanoic acid, benzoic acid, 4-methoxy benzoic acid and mixtures thereof. Examples of salts of organic acid include corresponding salts of these organic acids that are formed preferably with sodium and potassium, more preferably with sodium, e.g. trisodium citrate.

[0039] The composition preferably comprises an organic acid or its salts from 0.2 to 5 wt%, more preferably from 0.3 to 4 wt%, furthermore preferably from 0.4 to 3 wt%. The composition may optionally comprise ingredients, such as fragrance, colorant, foam boosting agents, and odor absorbing materials.

[0040] The composition of the invention is in liquid form. It comprises 60 to 99 wt% water, preferably from 85 to 95 wt% water. Composition preferably has a pH in the range of 4.0 to 9.0, preferably from 4.0 to 5.5. The pH of such a liquid composition is measured at 25 °C using a pH meter. The composition of the invention may be delivered as products which are specifically with the following application in mind.

[0041] General purpose and toilet cleaning composition:

[0042] The composition of the invention may be formulated as a general-purpose cleaner which is usually used to clean floors (in which case it may be known as a floor cleaner) or used to clean toilets (in which case it may be known as a toilet cleaner). It may also be used to clean other hard surfaces like furniture, tabletops, kitchen platforms and other surfaces in homes, offices, restaurants and other public places.

[0043] Ingredients like amphoteric surfactant and sequestrant may be included in the floor cleaner and toilet cleaner composition of the present invention.

[0044] General purpose cleaners and toilet cleaners may be diluted before use. When diluted, they may be diluted with water in a weight ratio in the range of 1 :10 to 1 :1000.

[0045] Kitchen cleaning composition:

[0046] The composition of the invention is useful for upkeep of areas in the kitchen e.g the sink, cleaning implement like sponge and scrub which may be cleaned using the dishwash composition, preferably in liquid form. Various ingredients other than the essential ingredients claimed in the present invention may be used as summarized below.

[0047] When formulated as a dishwashing composition, it may additionally include an anionic surfactant. Further preferably, the composition may comprise an amphoteric surfactant. Preferably, the amphoteric surfactants which may be used are the same as the ones listed under General purpose and toilet cleaning compositions. Amphoteric surfactant, when included, may be present in an amount ranging preferably from 0.1 to 5 wt%, more preferably from 0.1 to 4 wt%, even more preferably from 1 to 3 wt%.

[0048] Preferably, the composition may be used as is, i.e. neat, or it may be diluted before use. The extent of dilution is generally dependent on market choice. In some markets a more concentrated product is desired while in others a more diluted product is preferred. When the composition is liquid dishwash, it is typically diluted with water in a weight ratio between 1 :1 to 1 :10.

[0049] Another aspect of the present invention relates to a method of disrupting biofilm comprising the step of contacting the surface with a composition of the present invention preferably diluted with water. The surface may then be rinsed with enough water to deliver a surface substantially free of biofilm. The method is especially effective when the biofilm is mixed, comprising bacteria and fungi.

[0050] The invention will now be illustrated with the help of the following non-limiting examples. of mixed biofilm

[0051] Various compositions as shown in Table - 1 below were prepared and the disruption of mixed biofilm was measured using the procedure as given below:

[0052] Protocol for preparation of mixed biofilm containing bacteria and fungi

[0053] 1] For propagation of fungal and bacterial cells, Candida (in Sabouraud dextrose broth) and E. coli. (in Tryptic soya broth), were inoculated with a loopful of cells from the stock cultures and incubated overnight in an orbital shaker (150 - 180 rpm) at 30 °C. In the case of Aspergillus niger, conidia were harvested from 3-day old cultures on Sabouraud dextrose agar plates by flooding the surface of the plates with 5 ml of PBS containing 0.025%. (v / v) Tween-20 and rocked gently. The conidial suspension was then recovered and dispensed into a 15 ml sterile tube.

[0054] 2] The cells were harvested from the overnight grown liquid cultures (for Candida and E. coli) and the conidial suspension (for A. niger by centrifugation (approximately 3,000g for 5 min at 4 °C), supernatant was removed and washed twice in sterile PBS.

[0055] 3] The final pellet of cells was resuspended in approximately 20 ml of the appropriate medium that had been prewarmed to 37 °C: RPM I- 1640 for Candida and Aspergillus and TSB for E coli.

[0056] 4] From the resulting cell suspension 1:100 dilution was prepared in the same medium and counted using a haemocytometer.

[0057] 5] After counting, the volumes needed to prepare a suspension of cells was calculated.

[0058] Biofilm formation

[0059] 6] 96 well microtiter plates were used for the experiment, performing a minimum of 2 - 3 replicates.

[0060] 7] From the standardized inoculum prepared in step 5, 100 pl (for Candida and E coli) and 200 pl (for Aspergillus) was pipetted into selected wells of the microtiter plate(-s). 8] When all the selected wells had been seeded, the entire microtiter plate was covered with its original lid, sealed with parafilm, and placed inside an incubator and incubated statically for 7 days at 37 °C.

[0061] 9] Using a multichannel pipette the plates were washed three times in sterile PBS (200 - 300 pl per well) to remove planktonic and / or non-adherent cells that remain in the wells.

[0062] 10] Biofilms were now ready to be processed for susceptibility testing assays.

[0063] Preparation of test sample

[0064] 11] From the stock solution of each test sample to be tested, different concentrations were prepared using the same solvent.

[0065] 12] Using a multichannel pipette, 200 pl was added to the corresponding wells of each microtiter plate containing mixed biofilms, being careful not to touch or otherwise disrupt the biofilms.

[0066] 13] 100 pl of RPMI 1640 was added to each well in columns 2 to 10. 100 pl of RPMI 1640 was added to wells in column 11 , these will act as the positive control (biofilm not exposed to test sample). Wells in column 12 remain empty as negative controls.

[0067] 14] The contents were then mixed by gently pipetting up and down to perform a serial doubling dilution, and the pipette tips removed.

[0068] 15] The plates were covered with their lids, sealed with parafilm and incubated at 37 °C for different time intervals.

[0069] Preparation of XTT reagent

[0070] 16] The XTT is prepared as a saturated solution at 0.5 g / L in sterile Ringer’s lactate. Ringer’s lactate can be substituted for PBS or any other physiological buffer, as these have been shown to give similar reproducible results. The XTT solution is light sensitive, so it should be covered with aluminium foil during preparation. The solution needs to be filter-sterilized using a 0.22 pm- pore size filter; since it is a saturated solution, the filtration step will leave yellow residues on the filter, but this does not constitute a problem. Once prepared and filter-sterilized, aliquot into 10 ml working volumes, and stored at -70 °C.

[0071] 17] A 10 mM stock solution was prepared of menadione in 100% acetone. Aliquoted into smaller volumes (about 50 pl) and stored at -70°C.

[0072] Post-processing and measurement of biofilm disruption

[0073] 18] Thaw tubes containing 10 ml of the XTT solution as required for the experimental design was prepared (one per plate). To each tube, 1 pl of the stock solution of menadione was added to achieve a final menadione concentration of 1 pM. 19] Using a multichannel pipette 100 pl of the XTT / menadione solution was added to each well containing a pre-washed biofilm as well as to negative control wells (for the measurement of background XTT-colorimetric levels).

[0074] 20] The plates were covered in aluminium foil and incubated in the dark for 2 - 3 h at 37 °C.

[0075] 21] The plates were uncovered. At this point, visual inspection of the plates typically demonstrates a gradient of orange colour. Using a multichannel pipette 75 - 80 pl of the resulting-coloured supernatant was removed from each well and transfered into the wells of a new microtiter plate.

[0076] 22] The plate(-s) were read in a microtiter plate reader at 490 nm.

[0077] 23] From the resulting colorimetric readings (measured as optical density) and after subtracting the corresponding values for negative controls (from wells in column containing XTT only) the percentage biofilm inhibition was calculated by the test samples in comparison to control.

[0078] Percentage biofilm disruption was calculated by using the formula given below:

[0079] (Control - Treated)

[0080] - x 100

[0081] Control

[0082] Table - 1

[0083] In the above table, Lialet refers to Lialet 111-10 which is a fatty alcohol ethoxylate, a non-ionic surfactant having the chemical name C -Cn Alcohol Ethoxylate 10 EO.

[0084] Pectin used was GENU Pectin 105 Rapid set from CP Kelco, UK. The sample used had a degree of methyl esterification of 60%.

[0085] Fibersol used was sourced from ADM.

[0086] The bacteriophage used was M13 sourced from ATCC which targets E. Coli 15669.

[0087] The data in the Table - 1 above indicates that a composition as per the invention comprising pectin, bacteriophage and a surfactant (Example - 1) delivers superior biofilm disruption in short time frames (of 8 hours) as compared to subset combinations which achieve lower disruption even after a long contact time of 24 hours (Examples A, B, F). Further it is seen that inclusion of two polysaccharides (pectin and fibersol) in the composition of the invention can achieve much higher biofilm disruption in short times frames (8 hours) at even lower bacteriophage concentrations (Examples 2 and 3 as compared to Examples A-C, E, F).

[0088] Examples 4-12: Effect of using different types of surfactants:

[0089] Various combinations of the actives (Pectin + surfactant + bacteriophage) as per the invention using different surfactants were used and the resultant biofilm disruption was measured. The contact time in all of the examples was 8 hours. The compositions and the results are summarized in Table - 2:

[0090] Table - 2:

[0091] In the above table

[0092] SLES is an anionic surfactant and refers to sodium lauryl ether sulphate sourced from BASF. APG refers to alkyl poly glucoside which is a non-ionic surfactant sourced from BASF. The data in the above table indicates that for various concentrations of pectin and bacteriophage, a non-ionic surfactant (Examples 5, 6, 8, 9, 11 , 12) is superior to an anionic surfactant (Examples 4, 7,10). Within the non-ionic category, a fatty alcohol ethoxylate is superior (Examples 6, 9, 12) as compared to Examples (5, 8, and 11). Examples 13-21 : Effect of different surfactants when fibersol is used

[0093] Fibersol was used instead of Pectin in the compositions of Table - 2 and the experiments were repeated. The contact time in all of the examples was 8 hours. The data is summarized in Table - 3:

[0094] Table - 3:

[0095] The data in the above table indicates that similar trend with respect to type of surfactant is observed when fibersol is used instead of pectin viz. that non-ionic surfactant is superior to anionic surfactant and in non-ionic surfactant, a fatty alcohol ethoxylate is superior in delivering mixed biofilm disruption.

Claims

Claims1. A liquid composition for disruption of mixed biofilm comprising bacteria and fungus comprising(i) Bacteriophage capable of lysing bacteria selected from one or more of M. morganii, C. koseri, P. mirabilis, P. vulgaris and E. coir,(ii) polysaccharide selected from one or both of pectin and resistant dextrin;(iii) 0.1 to 25 wt% surfactant; and(iv) 60 to 99% water; wherein the composition has a pH in the range of 4.0 to 10.0.

2. A composition as claimed in claim 1 comprising bacteriophages capable of lysing bacteria C. koseri and E. Coli.

3. A composition as claimed in claim 1 comprising a mixture of bacteriophages capable of lysing bacteria M. morganii, C. koseri, P. mirabilis, P. vulgaris and E. coli.

4. A composition as claimed in any one of the preceding claims comprising 102to 1011PFU / ml bacteriophage.

5. A composition as claimed in claim in any one of the preceding claims wherein the biofilm is a mixed biofilm comprising bacteria and fungi.

6. A composition as claimed in any one of the preceding claims comprising 0.01 to 20 wt% polysaccharide.

7. A composition as claimed in any one of the preceding claims comprising pectin and resistant dextrin.

8. A composition as claimed in any one of the preceding claims wherein the surfactant is anionic or non-ionic.

9. A composition as claimed in claim 8 wherein the surfactant is non-ionic.

10. A composition as claimed in any one of the preceding claims comprising 1 to 10 wt% surfactant.

11. A method of disrupting mixed biofilm comprising bacteria and fungus comprising the step of contacting the surface with a composition as claimed in any one of the preceding claims, preferably diluted with water.

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