WO2019122470A1 - Disinfectant composition - Google Patents
Disinfectant composition Download PDFInfo
- Publication number
- WO2019122470A1 WO2019122470A1 PCT/ES2018/070785 ES2018070785W WO2019122470A1 WO 2019122470 A1 WO2019122470 A1 WO 2019122470A1 ES 2018070785 W ES2018070785 W ES 2018070785W WO 2019122470 A1 WO2019122470 A1 WO 2019122470A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- composition
- cect
- biofilm
- minutes
- lactic acid
- Prior art date
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- 239000000203 mixture Substances 0.000 title claims abstract description 122
- 239000000645 desinfectant Substances 0.000 title claims abstract description 14
- 239000003899 bactericide agent Substances 0.000 claims abstract description 24
- 231100000331 toxic Toxicity 0.000 claims abstract description 18
- 230000002588 toxic effect Effects 0.000 claims abstract description 18
- 229910001385 heavy metal Inorganic materials 0.000 claims abstract description 11
- 208000035143 Bacterial infection Diseases 0.000 claims abstract description 6
- 208000022362 bacterial infectious disease Diseases 0.000 claims abstract description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 64
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 50
- 241000894006 Bacteria Species 0.000 claims description 41
- 239000004310 lactic acid Substances 0.000 claims description 32
- 235000014655 lactic acid Nutrition 0.000 claims description 32
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 23
- 241000193755 Bacillus cereus Species 0.000 claims description 14
- 230000008030 elimination Effects 0.000 claims description 13
- 238000003379 elimination reaction Methods 0.000 claims description 13
- 238000011282 treatment Methods 0.000 claims description 13
- 241000194032 Enterococcus faecalis Species 0.000 claims description 12
- 241000588724 Escherichia coli Species 0.000 claims description 12
- 241000186779 Listeria monocytogenes Species 0.000 claims description 12
- KFSLWBXXFJQRDL-UHFFFAOYSA-N Peracetic acid Chemical compound CC(=O)OO KFSLWBXXFJQRDL-UHFFFAOYSA-N 0.000 claims description 12
- 241001354013 Salmonella enterica subsp. enterica serovar Enteritidis Species 0.000 claims description 9
- 229940032049 enterococcus faecalis Drugs 0.000 claims description 9
- 241000191967 Staphylococcus aureus Species 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 7
- 150000001299 aldehydes Chemical class 0.000 claims description 6
- 150000002989 phenols Chemical class 0.000 claims description 6
- 108010077895 Sarcosine Proteins 0.000 claims description 5
- 229960001927 cetylpyridinium chloride Drugs 0.000 claims description 5
- YMKDRGPMQRFJGP-UHFFFAOYSA-M cetylpyridinium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+]1=CC=CC=C1 YMKDRGPMQRFJGP-UHFFFAOYSA-M 0.000 claims description 5
- 239000003599 detergent Substances 0.000 claims description 5
- QBYIENPQHBMVBV-HFEGYEGKSA-N (2R)-2-hydroxy-2-phenylacetic acid Chemical compound O[C@@H](C(O)=O)c1ccccc1.O[C@@H](C(O)=O)c1ccccc1 QBYIENPQHBMVBV-HFEGYEGKSA-N 0.000 claims description 4
- IWYDHOAUDWTVEP-UHFFFAOYSA-N R-2-phenyl-2-hydroxyacetic acid Natural products OC(=O)C(O)C1=CC=CC=C1 IWYDHOAUDWTVEP-UHFFFAOYSA-N 0.000 claims description 4
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 4
- 229960002510 mandelic acid Drugs 0.000 claims description 4
- ZIWRUEGECALFST-UHFFFAOYSA-M sodium 4-(4-dodecoxysulfonylphenoxy)benzenesulfonate Chemical compound [Na+].CCCCCCCCCCCCOS(=O)(=O)c1ccc(Oc2ccc(cc2)S([O-])(=O)=O)cc1 ZIWRUEGECALFST-UHFFFAOYSA-M 0.000 claims description 4
- ZUFONQSOSYEWCN-UHFFFAOYSA-M sodium;2-(methylamino)acetate Chemical compound [Na+].CNCC([O-])=O ZUFONQSOSYEWCN-UHFFFAOYSA-M 0.000 claims description 4
- 231100000566 intoxication Toxicity 0.000 claims description 3
- 230000035987 intoxication Effects 0.000 claims description 3
- 229940079862 sodium lauryl sarcosinate Drugs 0.000 claims description 3
- ADWNFGORSPBALY-UHFFFAOYSA-M sodium;2-[dodecyl(methyl)amino]acetate Chemical compound [Na+].CCCCCCCCCCCCN(C)CC([O-])=O ADWNFGORSPBALY-UHFFFAOYSA-M 0.000 claims description 3
- 229910052793 cadmium Inorganic materials 0.000 claims description 2
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 abstract description 17
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- 238000011161 development Methods 0.000 description 8
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 8
- 244000005700 microbiome Species 0.000 description 8
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- 239000003242 anti bacterial agent Substances 0.000 description 4
- 229940088710 antibiotic agent Drugs 0.000 description 4
- 239000003139 biocide Substances 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- 231100000167 toxic agent Toxicity 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000032770 biofilm formation Effects 0.000 description 3
- 239000003344 environmental pollutant Substances 0.000 description 3
- KHFDEVIBCVSUAD-UHFFFAOYSA-N ethaneperoxoic acid Chemical compound CC(=O)OO.CC(=O)OO KHFDEVIBCVSUAD-UHFFFAOYSA-N 0.000 description 3
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- FQTLCLSUCSAZDY-UHFFFAOYSA-N (+) E(S) nerolidol Natural products CC(C)=CCCC(C)=CCCC(C)(O)C=C FQTLCLSUCSAZDY-UHFFFAOYSA-N 0.000 description 1
- MHVJRKBZMUDEEV-APQLOABGSA-N (+)-Pimaric acid Chemical compound [C@H]1([C@](CCC2)(C)C(O)=O)[C@@]2(C)[C@H]2CC[C@](C=C)(C)C=C2CC1 MHVJRKBZMUDEEV-APQLOABGSA-N 0.000 description 1
- 108010006533 ATP-Binding Cassette Transporters Proteins 0.000 description 1
- 102000005416 ATP-Binding Cassette Transporters Human genes 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- -1 Alkyl dimethyl benzyl ammonium chloride Chemical compound 0.000 description 1
- TXNJAVCZNMSELK-UHFFFAOYSA-N CCCCCCCCCCCCOC(=O)CNC Chemical compound CCCCCCCCCCCCOC(=O)CNC TXNJAVCZNMSELK-UHFFFAOYSA-N 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
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- DBVJJBKOTRCVKF-UHFFFAOYSA-N Etidronic acid Chemical compound OP(=O)(O)C(O)(C)P(O)(O)=O DBVJJBKOTRCVKF-UHFFFAOYSA-N 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
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- FQTLCLSUCSAZDY-ATGUSINASA-N Nerolidol Chemical compound CC(C)=CCC\C(C)=C\CC[C@](C)(O)C=C FQTLCLSUCSAZDY-ATGUSINASA-N 0.000 description 1
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- 229940027983 antiseptic and disinfectant quaternary ammonium compound Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000010065 bacterial adhesion Effects 0.000 description 1
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- 229910001424 calcium ion Inorganic materials 0.000 description 1
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- SYELZBGXAIXKHU-UHFFFAOYSA-N dodecyldimethylamine N-oxide Chemical compound CCCCCCCCCCCC[N+](C)(C)[O-] SYELZBGXAIXKHU-UHFFFAOYSA-N 0.000 description 1
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- XMMDVXFQGOEOKH-UHFFFAOYSA-N n'-dodecylpropane-1,3-diamine Chemical compound CCCCCCCCCCCCNCCCN XMMDVXFQGOEOKH-UHFFFAOYSA-N 0.000 description 1
- KVBGVZZKJNLNJU-UHFFFAOYSA-N naphthalene-2-sulfonic acid Chemical compound C1=CC=CC2=CC(S(=O)(=O)O)=CC=C21 KVBGVZZKJNLNJU-UHFFFAOYSA-N 0.000 description 1
- WASNIKZYIWZQIP-AWEZNQCLSA-N nerolidol Natural products CC(=CCCC(=CCC[C@@H](O)C=C)C)C WASNIKZYIWZQIP-AWEZNQCLSA-N 0.000 description 1
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- 150000003856 quaternary ammonium compounds Chemical class 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/40—Peroxides
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/36—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
- A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
Definitions
- the present invention fits in the sector of the food industry, in the sanitary field or can even be used at the domestic level.
- the present invention refers to a disinfectant composition, without toxic bactericidal agents or contaminants of the environment, applicable in any type of utensil or surface exposed to bacterial contamination.
- the composition of the invention can also be used for the treatment of patients afflicted with bacterial infections and / or patients with poisonings caused by heavy metals.
- a biofilm, biofilm, bacterial carpet or microbial mat is an organized microbial ecosystem, consisting of one or several microorganisms associated with a living or inert surface, with functional characteristics and complex structures. This type of microbial conformation occurs when the cells adhere to a surface or substrate, forming a community, which is characterized by the excretion of a protective extracellular adhesive matrix.
- a biofilm can contain approximately 15% cells and 85% extracellular matrix.
- This matrix is usually made up of exopolysaccharides, which form channels through which water, enzymes, nutrients and waste circulate.
- the cells establish relationships and dependencies: they live, cooperate and communicate through chemical signals (quorum perception), which regulate the expression of genes differently in different parts of the community, such as a tissue in a multicellular organism.
- biofilms In addition to the clinical or health field, it is important to take into account the formation of biofilms on the surfaces and utensils used both domestically and in the food industry.
- biofilms associated with the surfaces used in the food industry pose a problem of great importance because said biofilms can come into contact with food, and can cause diseases in the consumer once such contaminated food is ingested.
- these biofilms can cause significant economic losses because contaminated foods are discarded to avoid diseases associated with their consumption.
- Disinfectant formulas should be based on components that do not create long-term resistance, as this would hinder the therapeutic treatments of infections with pathogens that are increasingly resistant and invincible.
- the problem of resistance to antibiotics is one of the most alarming in the European Community due to the deaths caused by multiresistant bacteria (approximately 25,000 people per year in Europe) and also by the high cost of therapeutic treatments.
- the disinfectant compositions present in the state of the art usually contain among their components surfactants and detergents that are toxic and polluting to the environment. Therefore, it is considered essential to develop a composition capable of eliminating and / or preventing the formation of biofilms of pathogenic microorganisms in an effective manner, but which at the same time is a composition that does not comprise toxic bactericidal agents or pollutants for the environment, and that it also does not include components responsible for generating microbial resistances.
- the achievement of this composition would help the effective elimination of these biofilm-forming pathogens on different surfaces, interrupting for example the process of dissemination and transmission of infectious agents both to health personnel and patients in the clinical setting and contamination of food in the food industry.
- the composition of the invention is characterized by not comprising toxic bactericidal agents.
- the composition of the invention does not comprise toxic bactericidal agents selected from the list: phenols, heavy metals, aldehydes and detergents. More specifically, the composition of the invention does not comprise toxic bactericidal agents selected from the list: mandelic acid, peracetic acid, SDS, cetylpyridinium chloride, Sodium Cocoyl Sarcosinate, Lauryl sarcosinate sodium and / or Sodium dodecyl diphenyl ether disulfonate.
- compositions comprising hydrogen peroxide, lactic acid and EDTA are exemplified (see examples 1 to 10).
- all of the compositions disclosed in WO / 1993/002973 comprise concentrations of said three different components to the present invention and, in addition, comprise toxic compounds such as: peracetic acid (peroxyacetic acid) and glacial acetic acid .
- peracetic acid peroxyacetic acid
- glacial acetic acid a toxic compounds
- peracetic acid peroxyacetic acid
- exposure to peracetic acid can cause irritation to the skin, eyes and respiratory system, and longer or longer term exposure can cause permanent damage to the lungs.
- the composition of the invention by not understanding none of these toxic compounds clearly differs from WO / 1993/002973. It is important to note that the disinfectant composition of the invention is used mainly in the food industry sector, in the sanitary field or can even be used domestically. Therefore, it is of vital importance that the composition does not comprise toxic agents such as those included in the compositions disclosed in WO / 1993/002973. Although on page 8 line 31 to page 9 line 3 of WO / 1993/002973 the exclusion of compounds such as phenols and aldehydes from the composition is mentioned, however, according to that same paragraph, the removal of phenols and aldehydes is carried out in order to increase the activity of acids such as peroxyacetic acid (peracetic acid). Therefore, it follows from this paragraph that peroxyacetic acid (peracetic acid) and / or glacial acetic acid are always present in the compositions disclosed in WO / 1993/002973, as evidenced in examples 1 to 10) .
- peroxyacetic acid peracetic
- Naphthalene-2-sulfonic acid it is an environmental and xenobiotic pollutant, it causes damage to the eyes and skin burns. Also, it can cause cancer. It is corrosive.
- Usic acid is related to severe hepatotoxicity and liver failure. Daily oral intake of 300-1350 mg over a period of weeks has caused severe hepatotoxicity in several people.
- HEDP has a strong corrosive effect on the skin and mucous membranes.
- Nerolidol may cause sensitization by skin contact. It is harmful since it can cause lung damage if swallowed. Irritating to eyes, respiratory tract and skin. Toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment.
- Aminopropyl dodecylamine causes severe burns. It is harmful: risk of serious health effects in case of prolonged exposure by ingestion.
- Lauryl dimethylamine oxide aspiration can cause upper airway irritation and respiratory distress, most often in young children. Corneal injury has been reported after ocular exposure.
- Alkyl dimethyl benzyl ammonium chloride it is extensively toxic, it produces eye injuries, etc.
- US2009312279 is directed to the elimination of individual bacteria [0022] unlike the present invention where the total elimination of the biofilms formed by a cocktail of bacteria is achieved. Furthermore, as shown in the examples of the present invention, the composition of the invention allows to eliminate spore-forming bacteria such as Bacillus cereus, however US2009312279 does not disclose the possible elimination of spore-forming bacteria. Thus, in the present invention, unlike US2009312279, Staphylococcus aureus and Escherichia coli are eliminated, as well as Bacillus cereus, Listeria monocytogenes, Enterococcus faecalis and Salmonella.
- composition of the invention a disinfectant composition comprising lactic acid, hydrogen peroxide and EDTA (hereinafter composition of the invention) on biofilms composed of Gram-positive or Gram-negative bacteria, and also on mixed biofilms formed was investigated for multiple species. These biofilms are very resistant to the different treatments applied due to the low diffusion of the antimicrobial agents in the matrix of exopolysaccharides produced by the same microorganisms.
- the composition of the invention allows to avoid the development of these biofilms and also to eliminate them once established on different surfaces.
- the composition of the invention allows the solubilization of the structure of the biofilm and the diffusion of the antimicrobial agents that act synergistically to destroy the microorganisms that make up said structure.
- the composition of the invention turns out to be very effective in the elimination of biofilms and is characterized as not containing toxic bactericidal agents or contaminants of the environment.
- the composition of the invention comprises natural components produced by lactic acid bacteria, bacteria with the GRAS status "Generally Recognized As Safe", with antimicrobial effect such as hydrogen peroxide and lactic acid, which qualifies it as a natural product with antimicrobial effects.
- the components included in the composition of the invention do not have a specific target and therefore no bacterial resistance is created after successive applications as it happens with the quaternary ammonium compounds (cetylpyridinium chloride for example). It is important to note that hydrogen peroxide degrades rapidly to oxygen and Water. Thus, this component does not pose any risk to the environment as a pollutant.
- lactic acid as an environmentally friendly natural ingredient is easily degraded in C0 2 , CO and CH 4 .
- composition of the invention comprises:
- Lactic acid is an antimicrobial agent with a broad spectrum of action that is used in the food industry for its acidifying rather than antimicrobial power.
- hydrogen peroxide it is a powerful oxidizing agent and a bactericidal agent that first of all weakens the integrity of the biofilm in order to exert its bactericidal action on the microorganisms embedded in said structure. Both antimicrobials act synergistically to eliminate all microorganisms.
- EDTA as chelating agent and inhibitor of efflux pumps.
- the EDTA acts with the objective of extracting ions from the membrane of both Gram-negative and Gram-positive bacteria and thus facilitate the bactericidal action of the bactericidal agents used. It also acts as an inhibitor of efflux pumps of antimicrobials (responsible for nonspecific resistance) and thus increases the susceptibility of bacteria to the antibacterial action of antimicrobials.
- the three components act synergistically at precise concentrations to eliminate all microorganisms including sporulated bacteria such as Bacillus cereus whose elimination is a challenge for the food industry due to the resistance of their spores to both physical and chemical treatments.
- the contact time with highly contaminated surfaces (10 7 -10 8 UCF / ml) should be at least 2 minutes, preferably between 5-15 minutes, to reduce more than 99% of the microbial population of the biofilm.
- Lactic acid and hydrogen peroxide are among the antimicrobial substances produced by lactic acid bacteria (BAL).
- Lactic acid as a single or predominant fermentation product in BAL, plays a crucial role in food preservation, where up to 8% is produced in the fermentation process.
- the antimicrobial effect of lactic acid is able to inhibit bacterial growth through the disruptive action in the cytoplasmic membrane that leads to the loss of motive power of the proton and to the filtration of intracellular ions and constituents of Gram-positive and Gram-negative bacteria .
- lactic acid is not considered to be associated with chronic health risks and does not represent any risk to the environment.
- the composition of the invention allows to solve three important problems associated with the disinfection of surfaces: i) it is capable of eliminating biofilms efficiently in very short times of at least 2 minutes, preferably between 5-15 minutes, ii) no contains toxic bactericidal agents used in most disinfecting agents due to its destabilizing power of the integrity of the biofilm but which at the same time are associated with a high degree of toxicity and contamination of the environment and iii) it is capable of inhibiting the pumps of efflux responsible for the resistance of bacteria to antimicrobial agents.
- By having only antimicrobial agents with non-specific targets, does not create resistance after repeated applications as for example with the quaternary ammonium biocides that are used in different formulations such as cetilpiridinio.
- the composition of the invention can be applied for the elimination of biofilms comprised by pathogenic microorganisms in the health sector, the food industry and even the home.
- the composition of the invention can be used to decontaminate surfaces where food is handled, produced or packaged.
- the machinery or instruments used in these environments can be disinfected.
- companies in the food industry, slaughterhouses and even food sales areas may have an interest in this type of product, especially when it comes to a product that lacks toxicity or danger to create microbial resistances.
- its use also allows to decontaminate surfaces and instruments to avoid the development of biofilms or eliminate them once established. Therefore, contagion and nosocomial infections that can originate in this environment would be avoided.
- composition of the invention offers a high disinfecting power, being a natural product, which makes it devoid of toxicity.
- the composition of the invention can also be used for the treatment of patients suffering from bacterial infections and / or intoxications caused by heavy metals.
- the first aspect of the present invention refers to a composition comprising hydrogen peroxide, lactic acid and EDTA, characterized by not comprising toxic bactericidal agents.
- the composition of the invention is characterized by not comprising phenols, heavy metals, aldehydes and detergents.
- the composition of the invention is characterized by not comprising: mandelic acid, peracetic acid, SDS, cetylpyridinium chloride, Sodium Cocoyl Sarcosinate, sodium lauryl sarcosinate, Sodium dodecyl diphenyl ether disulfonate.
- the composition of the invention comprises: at least 3-6% hydrogen peroxide, at least 2.2-4.4% lactic acid and at least 12.5-25 mM EDTA. In a preferred aspect, the composition of the invention comprises: 6% hydrogen peroxide, 4.25% lactic acid and 25 mM EDTA.
- the second aspect of the present invention relates to a composition
- a composition comprising bactericidal agents consisting exclusively of hydrogen peroxide and lactic acid, and an efflux pump inhibiting chelating agent for example EDTA.
- the composition of the invention comprises two bactericidal agents consisting of at least 3-6% hydrogen peroxide and at least 2.2-4.4% lactic acid and as an efflux pump inhibiting chelating agent at least 12.5 -25 mM EDTA.
- the composition of the invention comprises two bactericidal agents consisting of 6% hydrogen peroxide and 4.25% lactic acid and as a chelating agent inhibitor of the 25 mM EDTA efflux pump.
- the third aspect of the present invention relates to a composition
- a composition comprising at least two bactericidal agents consisting of hydrogen peroxide and lactic acid and EDTA as a chelating agent inhibitor of the efflux pump.
- the composition of the invention comprises at least two bactericidal agents consisting of at least 3-6% hydrogen peroxide and at least 2.2-4.4% lactic acid and as a chelating agent inhibitor of the efflux pump minus 12.5-25 mM EDTA.
- the composition of the invention comprises at least two bactericidal agents consisting of 6% hydrogen peroxide and 4.25% lactic acid and as a chelating agent inhibitor of the 25 mM EDTA efflux pump.
- the fourth aspect of the invention refers to an ex vivo method (outside the human or animal body) for the elimination of biofilms formed by at least one bacterium, in any utensil or surface, comprising the application of the composition of the invention above described.
- the composition of the invention is applied to the biofilm for at least 2 minutes, preferably between 5 and 15 minutes.
- the bacteria that make up the biofilm are selected from the group comprising: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 and Solmonello Enteritidis UJ3449.
- the fifth aspect of the present invention refers to the ex vivo use of the composition of the invention as a disinfectant.
- the invention relates to the use of the composition of the invention for the elimination of biofilms formed by at least one bacterium.
- the composition is applied to the biofilm for at least 2 minutes, preferably between 5 and 15 minutes.
- the bacteria that make up the biofilm are: Staphylococcus oureus CECT 4468, Hysteria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 and Salmonella Enteritidis UJ3449.
- the sixth aspect of the present invention refers to the composition of the invention to be used as a medicine, particularly in the treatment of patients afflicted with bacterial infections and / or intoxications caused by heavy metals.
- the bacterium is selected from the list comprising: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 and Salmonella Enteritidis UJ3449.
- the heavy metal is Cadmium.
- the composition of the invention comprises concentrations or quantities precisely established in order to achieve a synergistic effect between its components that leads to the achievement of a triple technical effect: i) elimination of the biofilm in very short times of at least 2 minutes, preferably between 5-15 minutes, ii) without generating toxicity and iii) inhibiting at the same time the efflux pumps responsible for the resistance of the bacteria to antimicrobial agents.
- concentrations or amounts precisely established to achieve said synergistic effect are: at least 3-6% hydrogen peroxide, at least 2.2-4.4% lactic acid and at least 12.5-25 mM EDTA.
- the composition of the invention comprises 6% hydrogen peroxide, 4.25% lactic acid and 25 mM EDTA.
- Disinfectant means a product that eliminates or prevents the establishment of microorganisms in general and bacteria in particular. The use of a disinfectant allows to limit or even completely eliminate the contamination caused by microorganisms.
- Bodeicidal toxic agent means any bactericidal agent that has been classified as toxic in the state of the art. Thus, it would be an agent capable of causing the death of bacteria but at the same time exhibiting a certain toxicity for humans and animals after exposure to said agent.
- Examples of toxic bactericidal agents are: phenols, heavy metals, aldehydes and detergents, particularly mandelic acid, peracetic acid, SDS, cetylpyridinium chloride, Sodium Cocoyl Sarcosinate, sodium lauryl sarcosinate, Sodium dodecyl diphenyl ether disulfonate.
- FIG. 1 Antibacterial activity of the composition of the invention in mono and multispecific biofilms (the cocktail of six bacteria: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 and Salmonella Enteritidis UJ3449) for 0 min (control), 5 minutes (T5), 10 minutes (TIO), 20 minutes (T20) and 30 minutes (T30) at room temperature as determined by the determination of the viable count (Logio CFU / ml ). On the Y axis the Logio CFU / ml and the X axis samples are illustrated.
- Staphylococcus aureus CECT 4468 Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553
- Figure 2 Visualization of the inactivation of the multispecific biofilm using the BacLight Uve / Dead viability kit (Invitrogen) after the treatment with the composition of the invention (100% v / v) for 0 minutes (A, Control), 2 minutes (B), 5 minutes (C) and 10 minutes (D) at room temperature and resuspension of the biofilm in PBS. All images were obtained using LEICA TCS SP5 II confocal microscope (objective x63) and zoom of 2.5 (A and D), 1.5 (C) and 1 (B).
- FIG. 1 Photos of confocal microscopy of biofilm of the cocktail of bacteria treated or not with the compound. Left (control, live cells), center (treatment for 5 minutes, large amount of dead cells) and right (treatment for 10 minutes, all dead cells).
- Figure 4 The composition of the invention added to a sub-inhibitory concentration (1/2 MIC) to the cocktail of bacteria in TSB allowed the inhibition of the expression of genes coding for EffAB efflux pumps (efrA and efrB genes) and NorE (the norE gene).
- C Control).
- T treated with 1 ⁇ 2 MIC of the composition of the invention.
- Y axis Normalized relative expression).
- Example 1 Strains and bacterial growth conditions.
- Staphylococcus aureus CECT 4468 Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 and Salmonella Enteritidis UJ3449 were used in the present invention.
- Strains were grown in Tryptone Soya Broth (TSB) (Fluka, Madrid, Spain) at 37 ° C for 24 hours. The cultures were maintained in 20% glycerol at -20 ° C and -80 ° C for short and long term storage, respectively.
- TLB Tryptone Soya Broth
- MIC minimum inhibitory concentration
- MMC minimum bactericidal concentration
- the plates were incubated at 37 ° C under aerobic conditions for 24 hours and bacterial growth was evaluated by the presence of turbidity.
- the wells that exhibited the absence of turbidity were subjected to viable count determination (CFU / ml) by sowing the samples (10 ml) on Tryptone Soy agar plates (TSA). Subsequently the plates were incubated at 37 ° C for 24 hours.
- MIC was defined as the lowest concentration of the composition of the invention that inhibits visible growth and MBC was defined as the lowest concentration of the composition of the invention killing the bacteria (99%).
- TSA Tryptone Soy agar plates
- composition of the invention to different bacterial strains (S. aureus CECT 4468, L. monocytogenes CECT 4032, E. faecalis S-47, B. cereus CECT 5148, E. coli CCUG 47553 and S. Enteritidis UJ3449) and the cocktail of all strains were tested in microtiter plates. Overnight, bacterial cultures grown in TSB broth at 37 ° C for 24 hours were diluted 1/10 (v / v) in fresh TSB broth and 20 ml were added to each well of the 96-well microtiter plates.
- the wells were completed with 180 ml of TSB broth supplemented with the composition of the invention at sub-MIC concentrations (1/2 MIC of the composition of the invention for each strain up to 20% of the composition of the invention, v / v).
- the controls without the composition of the invention consisted of 180 pl of TSB broth.
- the plates were incubated at 37 ° C under aerobic conditions for 24 hours and the wells were washed with phosphate buffered saline (PBS).
- PBS phosphate buffered saline
- the anti-biofilm activity of the composition of the invention was determined by staining the wells washed with 100 ml of 1% (w / v) violet crystal which were incubated at room temperature for 15 minutes.
- Example 4 Antimicrobial effect of the composition of the invention on the preformed biofilm.
- the biofilms were resuspended in 200 ml of PBS and then seeded in TSA. The plates were incubated at 37 ° C for 24 hours for the determination of the total number of CFU / ml.
- Example 5 Microscopic evaluation of the effect of the composition of the invention on the biofilm.
- the imaging of the biofilm treated with the composition of the invention was performed using LIVE / DEAD BacLight TM (Thermo Fisher Scientific, Waltham, MA, USA) and a confocal laser scanning microscope (LEICA TCS-SP5 II, Mannheim , Germany) equipped with Plan-Apochromat 63x / 1.4 objective. After culturing the biofilm in a microtiter plate (200 pl) as described above, some wells were not treated with the composition of the invention (control) and the others were treated with the composition of the invention for 5 and 10 minutes. minutes at room temperature, washed with sterile PBS and resuspended in 50 ml of PBS.
- Example 6 Effect of the sub-inhibitory concentration of the composition of the invention on the resistance to the biofilm.
- RNA extraction was performed from planktonic bacterial cultures and also from multispecies biofilms using Direct-zol TM RNA kit (Zymo Research, USA) according to the manufacturer's instructions.
- Example 7 Statistical analysis. All analyzes were performed in triplicate. The statistical analyzes were performed using the Excel 2007 program (Microsoft Corporation, Redmond, Washington, USA). To determine averages and standard deviations. The statistical evaluation of the inhibition of biofilm development was performed by analysis of variance (ANOVA) using the Statgraphics Centurion XVI software (Statpoint Technologie, Warrenton, Virginia, USA). The same software was used to perform the Shapiro-Wilk and Levene tests to verify the normality of the data and to perform the multiple Tukey bilateral contrast to determine the differences by pairs between the strains, where the level of significance was established in the value P of ⁇ 0.05.
- ANOVA analysis of variance
- Example 8 Antimicrobial activity of the composition of the invention in planktonic cultures.
- Table 1 shows the antimicrobial effect exerted by the composition of the invention on the growth of the planktonic cells of each bacterium and also on the cocktail of all the bacteria tested. Table 1 shows the determination of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the composition of the invention.
- MIC Minimum Inhibitory Concentration
- MBC Minimum Bactericidal Concentration
- Example 9 Inhibition of biofilm formation by the composition of the invention.
- Example 10 Evaluation of the antimicrobial effect of the composition of the invention in biofilms.
- Example 11 The composition of the invention inhibits the efflux pumps.
- the composition of the invention is capable of inhibiting efflux pumps that act as non-specific resistance mechanisms in bacteria (particularly EfrAB and NorE efflux pumps). This is demonstrated in the present invention by analysis of the expression of said genes in the cocktail of bacteria studied in the absence and presence of low concentrations of the compound (1/2 MIC).
- the composition of the invention added to a sub-inhibitory concentration (1/2 MIC) to the bacterial cocktail in TSB allowed the inhibition of the expression of genes coding for efflow pumps EfrAB (efrA and efrB genes) and NorE (the norE gene) as shown in Figure 4. This Inhibition of efflux pumps reduces the spread of pathogens resistant to different antimicrobials. Thus, the development of biofilms with greater resistance to antimicrobials is avoided.
- the composition of the invention allows to potentiate the antimicrobial action of the two bactericidal agents (lactic acid and hydrogen peroxide) by blocking one of the defense mechanisms bacterial, thus allowing to restore the antimicrobial activity of various antimicrobial compounds that have lost their activity due to the presence of efflux pumps.
- the use of the composition of the invention by blocking or inhibiting the efflux pumps, will reduce the existing emergency relative to the existence of multiresistant bacteria to the antimicrobials.
- efflux pumps represent very attractive targets to reduce the spread of resistance to antimicrobials (antibiotics and biocides).
- the mechanism by which EDTA is believed to promote the inhibition of the efflux pump is by sequestering the calcium ions, thus, the hydrolysis of the ATP can not occur and the export pumps can not be put into operation.
- the family of ABC transporters as is the case with the EfrAB export pump.
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Abstract
The present invention can be used in the food industry and the health sector and can even be used in the home. Specifically, the invention relates to a disinfectant composition, without toxic or environmentally polluting bactericidal agents, which can be used on any type of utensil or surface exposed to bacterial contamination. The composition of the invention can also be used to treat patients suffering from bacterial infections and/or poisoning caused by heavy metals.
Description
COMPOSICIÓN DESINFECTANTE DISINFECTANT COMPOSITION
CAMPO DE LA INVENCIÓN FIELD OF THE INVENTION
La presente invención se encuadra en el sector de la industria alimentaria, en el ámbito sanitario o incluso puede ser usada a nivel doméstico. Concretamente, la presente invención hace referencia a una composición desinfectante, sin agentes bactericidas tóxicos o contaminantes del medio ambiente, aplicable en cualquier tipo de utensilio o superficie expuesta a la contaminación bacteriana. La composición de la invención también puede ser usada para el tratamiento de pacientes aquejados de infecciones bacterianas y/o de pacientes con intoxicaciones causadas por metales pesados. The present invention fits in the sector of the food industry, in the sanitary field or can even be used at the domestic level. Specifically, the present invention refers to a disinfectant composition, without toxic bactericidal agents or contaminants of the environment, applicable in any type of utensil or surface exposed to bacterial contamination. The composition of the invention can also be used for the treatment of patients afflicted with bacterial infections and / or patients with poisonings caused by heavy metals.
ESTADO DE LA TÉCNICA STATE OF THE ART
Una biopelícula, biofilm, tapiz bacteriano o tapete microbiano es un ecosistema microbiano organizado, conformado por uno o varios microorganismos asociados a una superficie viva o inerte, con características funcionales y estructuras complejas. Este tipo de conformación microbiana ocurre cuando las células se adhieren a una superficie o sustrato, formando una comunidad, que se caracteriza por la excreción de una matriz extracelular adhesiva protectora. A biofilm, biofilm, bacterial carpet or microbial mat is an organized microbial ecosystem, consisting of one or several microorganisms associated with a living or inert surface, with functional characteristics and complex structures. This type of microbial conformation occurs when the cells adhere to a surface or substrate, forming a community, which is characterized by the excretion of a protective extracellular adhesive matrix.
Una biopelícula puede contener aproximadamente un 15% de células y un 85% de matriz extracelular. Esta matriz generalmente está formada de exopolisacáridos, que forman canales por donde circulan agua, enzimas, nutrientes y residuos. Allí las células establecen relaciones y dependencias: viven, cooperan y se comunican a través de señales químicas (percepción de quorum), que regulan la expresión de genes de manera diferente en las distintas partes de la comunidad, como un tejido en un organismo multicelular. A biofilm can contain approximately 15% cells and 85% extracellular matrix. This matrix is usually made up of exopolysaccharides, which form channels through which water, enzymes, nutrients and waste circulate. There the cells establish relationships and dependencies: they live, cooperate and communicate through chemical signals (quorum perception), which regulate the expression of genes differently in different parts of the community, such as a tissue in a multicellular organism.
Se ha encontrado que más del 60% de las infecciones bacterianas son causadas por biopelículas. Por este motivo, han sido ampliamente estudiadas y se consideran una
amenaza clínica contundente ya que son capaces de crecer en catéteres e utensilios médicos y quirúrgicos. En el ámbito sanitario es donde hay más susceptibilidad a infecciones nosocomiales. It has been found that more than 60% of bacterial infections are caused by biofilms. For this reason, they have been widely studied and are considered a overwhelming clinical threat as they are able to grow in catheters and medical and surgical tools. In the health field, it is where there is more susceptibility to nosocomial infections.
Además del ámbito clínico o sanitario, es importante tener en cuenta la formación de biopelículas en las superficies y utensilios usados tanto a nivel doméstico como en la industria alimentaria. Particularmente, las biopelículas asociadas a las superficies usadas en la industria alimentaria, suponen un problema de mucha relevancia porque dichas biopelículas pueden llegar a estar en contacto con los alimentos, pudiendo causar enfermedades en el consumidor una vez ingeridos dichos alimentos contaminados. Además, dichas biopelículas pueden causar pérdidas económicas importantes debido a que los alimentos contaminados son desechados para evitar enfermedades asociadas a su consumo. In addition to the clinical or health field, it is important to take into account the formation of biofilms on the surfaces and utensils used both domestically and in the food industry. In particular, the biofilms associated with the surfaces used in the food industry pose a problem of great importance because said biofilms can come into contact with food, and can cause diseases in the consumer once such contaminated food is ingested. In addition, these biofilms can cause significant economic losses because contaminated foods are discarded to avoid diseases associated with their consumption.
Por otro lado, es importante tener en cuenta los problemas asociados con el incremento de la resistencia a antimicrobianos y la resistencia cruzada entre los antibióticos (usados en terapia) y biocidas (usados como desinfectantes). Las fórmulas de los desinfectantes deberían estar basadas en componentes que no creen resistencia a largo plazo, ya que esto dificultaría los tratamientos terapéuticos de las infecciones con patógenos que cada vez son más resistentes e invencibles. Actualmente, el problema de resistencia a antibióticos es uno de los más alarmantes en la Comunidad Europea debido a las muertes causadas por bacterias multirresistentes (aproximadamente 25000 personas por año en Europa) y también por el elevado coste de los tratamientos terapéuticos. On the other hand, it is important to take into account the problems associated with increased antimicrobial resistance and cross-resistance between antibiotics (used in therapy) and biocides (used as disinfectants). Disinfectant formulas should be based on components that do not create long-term resistance, as this would hinder the therapeutic treatments of infections with pathogens that are increasingly resistant and invincible. Currently, the problem of resistance to antibiotics is one of the most alarming in the European Community due to the deaths caused by multiresistant bacteria (approximately 25,000 people per year in Europe) and also by the high cost of therapeutic treatments.
En el estado de la técnica se ha descrito mucha información relativa a diversas composiciones que permiten eliminar las biopelículas. Sin embargo, las composiciones desinfectantes presentes en el estado de la técnica suelen contener entre sus componentes surfactantes y detergentes que son tóxicos y contaminantes para el medio ambiente.
Por lo tanto, se considera indispensable desarrollar una composición capaz de eliminar y/o evitar la formación de biopelículas de microorganismos patógenos de forma eficaz, pero que al mismo tiempo sea una composición que no comprenda agentes bactericidas tóxicos, ni contaminantes para el medio ambiente, y que tampoco comprenda componentes responsables de generar resistencias microbianas. La consecución de esta composición ayudaría a la eliminación eficaz de estos patógenos formadores de biopelículas en las diferentes superficies, interrumpiendo por ejemplo el proceso de diseminación y de transmisión de los agentes infecciosos tanto al personal sanitario como a los pacientes en el ámbito clínico y la contaminación de los alimentos en la industria alimentaria. In the state of the art a lot of information has been described relative to various compositions that allow biofilms to be eliminated. However, the disinfectant compositions present in the state of the art usually contain among their components surfactants and detergents that are toxic and polluting to the environment. Therefore, it is considered essential to develop a composition capable of eliminating and / or preventing the formation of biofilms of pathogenic microorganisms in an effective manner, but which at the same time is a composition that does not comprise toxic bactericidal agents or pollutants for the environment, and that it also does not include components responsible for generating microbial resistances. The achievement of this composition would help the effective elimination of these biofilm-forming pathogens on different surfaces, interrupting for example the process of dissemination and transmission of infectious agents both to health personnel and patients in the clinical setting and contamination of food in the food industry.
Por lo tanto, a la hora de evaluar el estado de la técnica, es muy importante considerar que la composición de la invención se caracteriza por no comprender agentes bactericidas tóxicos. Particularmente, la composición de la invención no comprende agentes bactericidas tóxicos seleccionados de la lista: fenoles, metales pesados, aldehidos y detergentes. Más específicamente, la composición de la invención no comprende agentes bactericidas tóxicos seleccionados de la lista: ácido mandélico, ácido peracético, SDS, cloruro de cetilpiridinio, Sodio Cocoyl Sarcosinato, Lauril sarcosinato de sodio y/o Sodio dodecil difenil éter disulfonato. Therefore, when evaluating the state of the art, it is very important to consider that the composition of the invention is characterized by not comprising toxic bactericidal agents. Particularly, the composition of the invention does not comprise toxic bactericidal agents selected from the list: phenols, heavy metals, aldehydes and detergents. More specifically, the composition of the invention does not comprise toxic bactericidal agents selected from the list: mandelic acid, peracetic acid, SDS, cetylpyridinium chloride, Sodium Cocoyl Sarcosinate, Lauryl sarcosinate sodium and / or Sodium dodecyl diphenyl ether disulfonate.
En este sentido, se menciona como estado de la técnica al documento WO/1993/002973 donde se ejemplifican (ver ejemplos 1 a 10) composiciones que comprenden peróxido de hidrogeno, ácido láctico y EDTA. Sin embargo, es importante hacer notar que todas las composiciones divulgadas en WO/1993/002973 comprenden concentraciones de dichos tres componentes diferentes a la presente invención y, además, comprenden compuestos tóxicos tales como: ácido peracético (ácido ácido peroxiacético) y ácido acético glacial. En particular, la exposición al ácido peracético puede causar irritación en la piel, ojos y sistema respiratorio, y una exposición mayor o a largo plazo puede causar daño permanente a los pulmones. Además, ha habido casos de asma ocupacional causados por el ácido peracético. Por lo tanto, la composición de la invención, al no comprender
ninguno de estos compuestos tóxicos, se diferencia claramente del documento WO/1993/002973. Es importante hacer notar que la composición desinfectante de la invención se utiliza fundamentalmente en el sector de la industria alimentaria, en el ámbito sanitario o incluso puede ser usada a nivel doméstico. Por lo tanto, es de vital importancia que la composición no comprenda agentes tóxicos tales como los incluidos en las composiciones divulgadas en WO/1993/002973. Aunque en la página 8 línea 31 a página 9 línea 3 de WO/1993/002973 se menciona la exclusión de compuestos como fenoles y aldehidos de la composición, sin embargo, según ese mismo párrafo, la eliminación de fenoles y aldehidos se realiza para aumentar la actividad de ácidos tales como el ácido peroxiacético (ácido peracético). Por lo tanto, de este párrafo se deduce que el ácido peroxiacético (ácido peracético) y/o el ácido acético glacial están siempre presentes en las composiciones divulgadas en WO/1993/002973, tal y como se evidencia en los ejemplos 1 a 10). In this regard, document WO / 1993/002973 is mentioned as state of the art, where compositions comprising hydrogen peroxide, lactic acid and EDTA are exemplified (see examples 1 to 10). However, it is important to note that all of the compositions disclosed in WO / 1993/002973 comprise concentrations of said three different components to the present invention and, in addition, comprise toxic compounds such as: peracetic acid (peroxyacetic acid) and glacial acetic acid . In particular, exposure to peracetic acid can cause irritation to the skin, eyes and respiratory system, and longer or longer term exposure can cause permanent damage to the lungs. In addition, there have been cases of occupational asthma caused by peracetic acid. Therefore, the composition of the invention, by not understanding none of these toxic compounds clearly differs from WO / 1993/002973. It is important to note that the disinfectant composition of the invention is used mainly in the food industry sector, in the sanitary field or can even be used domestically. Therefore, it is of vital importance that the composition does not comprise toxic agents such as those included in the compositions disclosed in WO / 1993/002973. Although on page 8 line 31 to page 9 line 3 of WO / 1993/002973 the exclusion of compounds such as phenols and aldehydes from the composition is mentioned, however, according to that same paragraph, the removal of phenols and aldehydes is carried out in order to increase the activity of acids such as peroxyacetic acid (peracetic acid). Therefore, it follows from this paragraph that peroxyacetic acid (peracetic acid) and / or glacial acetic acid are always present in the compositions disclosed in WO / 1993/002973, as evidenced in examples 1 to 10) .
Por otro lado, también se menciona el documento US2009312279. En este documento, particularmente en la Tabla 1, se divulgan diferentes combinaciones de tres componentes. Sin embargo, US2009312279 no divulga los tres componentes peróxido de hidrógeno, ácido láctico y EDTA en combinación. Este documento divulga que sus composiciones pueden comprender ácido láctico entre otros ácidos, pero no divulga una combinación de dicho ácido láctico con peróxido de hidrógeno y EDTA. Por otro lado, debe de tenerse en cuenta que atendido a la Tabla 1 de US2009312279, las composiciones que tienen algún componente de los incluidos en la composición de la presente invención, van además acompañados de compuestos conocidos en el estado de la técnica por su toxicidad. A modo de ejemplo se identifican los siguientes: On the other hand, document US2009312279 is also mentioned. In this document, particularly in Table 1, different combinations of three components are disclosed. However, US2009312279 does not disclose the three components hydrogen peroxide, lactic acid and EDTA in combination. This document discloses that its compositions may comprise lactic acid among other acids, but does not disclose a combination of said lactic acid with hydrogen peroxide and EDTA. On the other hand, it must be taken into account that given the Table 1 of US2009312279, the compositions that have some component of those included in the composition of the present invention, are also accompanied by compounds known in the state of the art due to their toxicity . By way of example, the following are identified:
• Ácido naftaleno-2-sulfónico: es contaminante ambiental y xenobiótico, causa daños en los ojos y quemaduras en la piel. También, puede causar cáncer. Es corrosivo.
• Ácido úsnico: está relacionado con hepatotoxicidad grave e insuficiencia hepática. La ingesta oral diaria de 300-1350 mg durante un período de semanas ha provocado hepatotoxicidad grave en varias personas. • Naphthalene-2-sulfonic acid: it is an environmental and xenobiotic pollutant, it causes damage to the eyes and skin burns. Also, it can cause cancer. It is corrosive. • Usic acid: is related to severe hepatotoxicity and liver failure. Daily oral intake of 300-1350 mg over a period of weeks has caused severe hepatotoxicity in several people.
• HEDP: tiene un fuerte efecto corrosivo sobre la piel y las membranas mucosas. • HEDP: has a strong corrosive effect on the skin and mucous membranes.
• Nerolidol: puede provocar sensibilización por contacto con la piel. Es nocivo ya que puede causar daño pulmonar si se ingiere. Irritante para los ojos, las vías respiratorias y la piel. Tóxico para los organismos acuáticos, puede causar efectos adversos a largo plazo en el medio ambiente acuático. • Nerolidol: may cause sensitization by skin contact. It is harmful since it can cause lung damage if swallowed. Irritating to eyes, respiratory tract and skin. Toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment.
• Aminopropil dodecilamina: provoca quemaduras graves. Es nocivo: riesgo de efectos graves para la salud en caso de exposición prolongada por ingestión. • Aminopropyl dodecylamine: causes severe burns. It is harmful: risk of serious health effects in case of prolonged exposure by ingestion.
• Lauril dimetilamina óxido: la aspiración puede causar irritación de la vía aérea superior y dificultad respiratoria, más a menudo en niños pequeños. La lesión corneal se ha reportado después de la exposición ocular. • Lauryl dimethylamine oxide: aspiration can cause upper airway irritation and respiratory distress, most often in young children. Corneal injury has been reported after ocular exposure.
• 2-Hidroxipropil- -Ciclodextrina: puede ocasionar inflamación de los pulmones. • 2-Hydroxypropyl-Cyclodextrin: can cause inflammation of the lungs.
• Cloruro de alquil dimetil bencil amonio: es ampliamente tóxico, produce lesiones oculares, etc. • Alkyl dimethyl benzyl ammonium chloride: it is extensively toxic, it produces eye injuries, etc.
Por otro lado, US2009312279 se dirige a la eliminación de bacterias individuales [0022] a diferencia de la presente invención donde se consigue la eliminación total del biofilms formados por un cóctel de bacterias. Además, tal y como se muestra en los ejemplos de la presente invención, la composición de la invención permite eliminar bacterias formadoras de esporas tales como Bacillus cereus, sin embargo US2009312279 no divulga la posible eliminación de bacterias formadoras de esporas. Así, en la presente invención, a diferencia de US2009312279 se eliminan Staphylococcus aureus y Escherichia coli, así como Bacillus cereus, Listeria monocytogenes, Enterococcus faecalis y Salmonella. On the other hand, US2009312279 is directed to the elimination of individual bacteria [0022] unlike the present invention where the total elimination of the biofilms formed by a cocktail of bacteria is achieved. Furthermore, as shown in the examples of the present invention, the composition of the invention allows to eliminate spore-forming bacteria such as Bacillus cereus, however US2009312279 does not disclose the possible elimination of spore-forming bacteria. Thus, in the present invention, unlike US2009312279, Staphylococcus aureus and Escherichia coli are eliminated, as well as Bacillus cereus, Listeria monocytogenes, Enterococcus faecalis and Salmonella.
Por lo tanto, se puede concluir que no se ha localizado ningún documento del estado de la técnica que divulgue una composición que i) comprenda peróxido de hidrógeno, ácido láctico y EDTA (particularmente a las concentraciones usadas en la presente invención y ii) que no incluya compuestos bactericidas tóxicos. Además, no se ha localizado ningún
documento en el estado de la técnica donde se divulgue la composición de la invención para la eliminación completa de biofilms formados por un cóctel de bacterias que comprende, entre otras, bacterias formadoras de esporas. Therefore, it can be concluded that no prior art document has been located that discloses a composition that i) comprises hydrogen peroxide, lactic acid and EDTA (particularly at the concentrations used in the present invention and ii) that does not include toxic bactericidal compounds. In addition, no document in the state of the art where the composition of the invention is disclosed for the complete elimination of biofilms formed by a cocktail of bacteria comprising, among others, spore-forming bacteria.
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
Breve descripción de la invención BRIEF DESCRIPTION OF THE INVENTION
En la presente invención, se investigó el efecto de una composición desinfectante que comprende ácido láctico, peróxido de hidrógeno y EDTA (en adelante composición de la invención) sobre biopelículas compuestas por bacterias Gram-positivas o Gram-negativas, y también sobre biopelículas mixtas formadas por múltiples especies. Estas biopelículas son muy resistentes a los diferentes tratamientos aplicados debido a la baja difusión de los agentes antimicrobianos en la matriz de exopolisacáridos producidos por los mismos microorganismos. La composición de la invención permite evitar el desarrollo de estas biopelículas y también eliminarlas una vez establecidas sobre diferentes superficies. La composición de la invención permite la solubilización de la estructura de la biopelícula y la difusión de los agentes antimicrobianos que actúan sinérgicamente para destruir los microorganismos que componen dicha estructura. In the present invention, the effect of a disinfectant composition comprising lactic acid, hydrogen peroxide and EDTA (hereinafter composition of the invention) on biofilms composed of Gram-positive or Gram-negative bacteria, and also on mixed biofilms formed was investigated for multiple species. These biofilms are very resistant to the different treatments applied due to the low diffusion of the antimicrobial agents in the matrix of exopolysaccharides produced by the same microorganisms. The composition of the invention allows to avoid the development of these biofilms and also to eliminate them once established on different surfaces. The composition of the invention allows the solubilization of the structure of the biofilm and the diffusion of the antimicrobial agents that act synergistically to destroy the microorganisms that make up said structure.
La composición de la invención resulta ser muy eficaz en la eliminación de biopelículas y se caracteriza por no contener agentes bactericidas tóxicos ni contaminantes del medio ambiente. La composición de la invención comprende componentes naturales producidos por las bacterias del ácido láctico, bacterias con el estatus GRAS "Generally Recognized As Safe", con efecto antimicrobiano tales como el peróxido de hidrógeno y el ácido láctico, lo cual lo califica como producto natural con efectos antimicrobianos. Por otro lado, los componentes incluidos en la composición de la invención no tienen una diana específica y por lo tanto no se crean resistencias bacterianas tras sucesivas aplicaciones tal como sí ocurre con los compuestos de amonio cuaternario (cloruro de cetilpiridinio por ejemplo). Es importante indicar que el peróxido de hidrógeno se degrada rápidamente a oxígeno y
agua. Así, este componente no supone ningún riesgo para el medio ambiente como contaminante. Por su parte, el ácido láctico como ingrediente natural respetuoso con el medio ambiente se degrada fácilmente en C02, CO y CH4. The composition of the invention turns out to be very effective in the elimination of biofilms and is characterized as not containing toxic bactericidal agents or contaminants of the environment. The composition of the invention comprises natural components produced by lactic acid bacteria, bacteria with the GRAS status "Generally Recognized As Safe", with antimicrobial effect such as hydrogen peroxide and lactic acid, which qualifies it as a natural product with antimicrobial effects. On the other hand, the components included in the composition of the invention do not have a specific target and therefore no bacterial resistance is created after successive applications as it happens with the quaternary ammonium compounds (cetylpyridinium chloride for example). It is important to note that hydrogen peroxide degrades rapidly to oxygen and Water. Thus, this component does not pose any risk to the environment as a pollutant. For its part, lactic acid as an environmentally friendly natural ingredient is easily degraded in C0 2 , CO and CH 4 .
Particularmente la composición de la invención comprende: Particularly, the composition of the invention comprises:
• Dos agentes bactericidas: el ácido láctico y el peróxido de hidrógeno. El ácido láctico es un agente antimicrobiano con un amplio espectro de acción que se usa en la industria alimentaria por su poder acidulante más que antimicrobiano. En cuanto al peróxido de hidrógeno es un potente agente oxidante y un agente bactericida que permite en primer lugar debilitar la integridad de la biopelícula para así ejercer su acción bactericida sobre los microorganismos embebidos en dicha estructura. Ambos antimicrobianos actúan sinérgicamente para eliminar todos los microorganismos. • Two bactericidal agents: lactic acid and hydrogen peroxide. Lactic acid is an antimicrobial agent with a broad spectrum of action that is used in the food industry for its acidifying rather than antimicrobial power. As for hydrogen peroxide, it is a powerful oxidizing agent and a bactericidal agent that first of all weakens the integrity of the biofilm in order to exert its bactericidal action on the microorganisms embedded in said structure. Both antimicrobials act synergistically to eliminate all microorganisms.
• EDTA como agente quelante e inhibidor de las bombas de eflujo. El EDTA actúa con el objetivo de extraer iones de la membrana de las bacterias tanto Gram- negativas como Gram-positivas y así facilitar la acción bactericida de los agentes bactericidas empleados. Además actúa como inhibidor de bombas de eflujo de los antimicrobianos (responsables de la resistencia inespecífica) y así incrementa la susceptibilidad de las bacterias a la acción bactericida de los antimicrobianos. • EDTA as chelating agent and inhibitor of efflux pumps. The EDTA acts with the objective of extracting ions from the membrane of both Gram-negative and Gram-positive bacteria and thus facilitate the bactericidal action of the bactericidal agents used. It also acts as an inhibitor of efflux pumps of antimicrobials (responsible for nonspecific resistance) and thus increases the susceptibility of bacteria to the antibacterial action of antimicrobials.
Los tres componentes actúan sinérgicamente a concentraciones precisas para eliminar todos los microorganismos incluso las bacterias esporuladas tales como Bacillus cereus cuya eliminación es un reto para la industria alimentaria debido a la resistencia de sus esporas tanto a tratamientos físicos como químicos. The three components act synergistically at precise concentrations to eliminate all microorganisms including sporulated bacteria such as Bacillus cereus whose elimination is a challenge for the food industry due to the resistance of their spores to both physical and chemical treatments.
El tiempo de contacto con las superficies altamente contaminadas (107-108 UCF/ml) debe ser al menos de 2 minutos, preferentemente entre 5-15 minutos, para reducir más del 99% de la población microbiana de la biopelícula. El ácido láctico y el peróxido de hidrógeno se encuentran entre las sustancias antimicrobianas producidas por las
bacterias del ácido láctico (BAL). El ácido láctico, como producto de fermentación único o predominante en las BAL, desempeña un papel crucial en la conservación de alimentos, donde se produce hasta en un 8% en el proceso de fermentación. El efecto antimicrobiano del ácido láctico es capaz de inhibir el crecimiento bacteriano a través de la acción disruptiva en la membrana citoplasmática que conduce a la pérdida de fuerza motriz del protón y a la filtración de iones intracelulares y constituyentes de bacterias Gram-positivas y Gram-negativas. Además, desde el punto de vista de la seguridad, no se considera que el ácido láctico esté asociado a riesgos crónicos para la salud y no represente ningún riesgo para el medio ambiente. The contact time with highly contaminated surfaces (10 7 -10 8 UCF / ml) should be at least 2 minutes, preferably between 5-15 minutes, to reduce more than 99% of the microbial population of the biofilm. Lactic acid and hydrogen peroxide are among the antimicrobial substances produced by lactic acid bacteria (BAL). Lactic acid, as a single or predominant fermentation product in BAL, plays a crucial role in food preservation, where up to 8% is produced in the fermentation process. The antimicrobial effect of lactic acid is able to inhibit bacterial growth through the disruptive action in the cytoplasmic membrane that leads to the loss of motive power of the proton and to the filtration of intracellular ions and constituents of Gram-positive and Gram-negative bacteria . Furthermore, from the point of view of safety, lactic acid is not considered to be associated with chronic health risks and does not represent any risk to the environment.
Así, la composición de la invención permite resolver tres importantes problemas asociados con la desinfección de superficies: i) es capaz de eliminar las biopelículas de forma eficaz en tiempos muy cortos de al menos 2 minutos, preferentemente entre 5-15 minutos, ii) no contiene agentes bactericidas tóxicos usados en la mayoría de los agentes desinfectantes debido a su poder desestabilizante de la integridad de la biopelícula pero que al mismo tiempo están asociados con un alto grado de toxicidad y contaminación del medio ambiente y iii) es capaz de inhibir las bombas de eflujo responsables de la resistencia de las bacterias a agentes antimicrobianos. Al tener solo agentes antimicrobianos con dianas no específicas, no crea resistencia tras repetidas aplicaciones como ocurre por ejemplo con los biocidas de amonio cuaternario que se usan en las diferentes formulaciones tales como cetilpiridinio. Thus, the composition of the invention allows to solve three important problems associated with the disinfection of surfaces: i) it is capable of eliminating biofilms efficiently in very short times of at least 2 minutes, preferably between 5-15 minutes, ii) no contains toxic bactericidal agents used in most disinfecting agents due to its destabilizing power of the integrity of the biofilm but which at the same time are associated with a high degree of toxicity and contamination of the environment and iii) it is capable of inhibiting the pumps of efflux responsible for the resistance of bacteria to antimicrobial agents. By having only antimicrobial agents with non-specific targets, does not create resistance after repeated applications as for example with the quaternary ammonium biocides that are used in different formulations such as cetilpiridinio.
Por lo tanto, la composición de la invención puede ser aplicada para la eliminación de biopelículas comprendidas por los microorganismos patógenos en el sector sanitario, la industria alimentaria e incluso el hogar. En el sector de la industria alimentaria, la composición de la invención se puede utilizar para descontaminar superficies donde se manipulan, se producen o se envasan alimentos. Además, se pueden desinfectar las maquinarias o instrumentos usados en estos ambientes. En este sentido, las empresas de la industria alimentaria, mataderos e incluso áreas de venta de los alimentos pueden tener interés en este tipo de productos especialmente cuando se trata de un producto
que carece de toxicidad o peligro para crear resistencias microbianas. En el sector sanitario o clínico, su uso también permite descontaminar superficies e instrumentos para evitar el desarrollo de biopelículas o eliminarlas una vez establecidas. Por lo tanto, se evitaría el contagio y las infecciones nosocomiales que se pueden originar en este ambiente. A nivel doméstico, es importante destacar que la composición de la invención ofrece un alto poder desinfectante siendo un producto natural, lo que hace que carezca de toxicidad. La composición de la invención también puede ser usada para el tratamiento de pacientes aquejados de infecciones bacterianas y/o de intoxicaciones causadas por metales pesados. Therefore, the composition of the invention can be applied for the elimination of biofilms comprised by pathogenic microorganisms in the health sector, the food industry and even the home. In the food industry sector, the composition of the invention can be used to decontaminate surfaces where food is handled, produced or packaged. In addition, the machinery or instruments used in these environments can be disinfected. In this sense, companies in the food industry, slaughterhouses and even food sales areas may have an interest in this type of product, especially when it comes to a product that lacks toxicity or danger to create microbial resistances. In the sanitary or clinical sector, its use also allows to decontaminate surfaces and instruments to avoid the development of biofilms or eliminate them once established. Therefore, contagion and nosocomial infections that can originate in this environment would be avoided. At the domestic level, it is important to emphasize that the composition of the invention offers a high disinfecting power, being a natural product, which makes it devoid of toxicity. The composition of the invention can also be used for the treatment of patients suffering from bacterial infections and / or intoxications caused by heavy metals.
Por lo tanto, el primer aspecto de la presente invención hace referencia a una composición que comprende peróxido de hidrógeno, ácido láctico y EDTA, caracterizada por no comprender agentes bactericidas tóxicos. En un aspecto preferido, la composición de la invención se caracteriza por no comprender fenoles, metales pesados, aldehidos y detergentes. En un aspecto preferido, la composición de la invención se caracteriza por no comprender: ácido mandélico, ácido peracético, SDS, cloruro de cetilpiridinio, Sodio Cocoyl Sarcosinato, Lauril sarcosinato de sodio, Sodio dodecil difenil éter disulfonato. En un aspecto preferido, la composición de la invención comprende: al menos 3-6% de peróxido de hidrógeno, al menos 2.2-4.4% de ácido láctico y al menos 12.5-25 mM de EDTA. En un aspecto preferido, la composición de la invención comprende: 6% de peróxido de hidrógeno, 4.25% de ácido láctico y 25 mM de EDTA. Therefore, the first aspect of the present invention refers to a composition comprising hydrogen peroxide, lactic acid and EDTA, characterized by not comprising toxic bactericidal agents. In a preferred aspect, the composition of the invention is characterized by not comprising phenols, heavy metals, aldehydes and detergents. In a preferred aspect, the composition of the invention is characterized by not comprising: mandelic acid, peracetic acid, SDS, cetylpyridinium chloride, Sodium Cocoyl Sarcosinate, sodium lauryl sarcosinate, Sodium dodecyl diphenyl ether disulfonate. In a preferred aspect, the composition of the invention comprises: at least 3-6% hydrogen peroxide, at least 2.2-4.4% lactic acid and at least 12.5-25 mM EDTA. In a preferred aspect, the composition of the invention comprises: 6% hydrogen peroxide, 4.25% lactic acid and 25 mM EDTA.
El segundo aspecto de la presente invención hace referencia a una composición que comprende agentes bactericidas que consisten exclusivamente en peróxido de hidrógeno y ácido láctico, y un agente quelante inhibidor de la bomba de eflujo por ejemplo el EDTA. En un aspecto preferido, la composición de la invención comprende dos agentes bactericidas que consisten en al menos 3-6% de peróxido de hidrógeno y al menos 2.2- 4.4% de ácido láctico y como agente quelante inhibidor de la bomba de eflujo al menos 12.5-25 mM de EDTA. En un aspecto preferido, la composición de la invención comprende
dos agentes bactericidas que consisten en 6% de peróxido de hidrógeno y 4.25% de ácido láctico y como agente quelante inhibidor de la bomba de eflujo 25 mM de EDTA. The second aspect of the present invention relates to a composition comprising bactericidal agents consisting exclusively of hydrogen peroxide and lactic acid, and an efflux pump inhibiting chelating agent for example EDTA. In a preferred aspect, the composition of the invention comprises two bactericidal agents consisting of at least 3-6% hydrogen peroxide and at least 2.2-4.4% lactic acid and as an efflux pump inhibiting chelating agent at least 12.5 -25 mM EDTA. In a preferred aspect, the composition of the invention comprises two bactericidal agents consisting of 6% hydrogen peroxide and 4.25% lactic acid and as a chelating agent inhibitor of the 25 mM EDTA efflux pump.
El tercer aspecto de la presente invención hace referencia a una composición que comprende al menos dos agentes bactericidas que consisten en peróxido de hidrógeno y ácido láctico y EDTA como agente quelante inhibidor de la bomba de eflujo. En un aspecto preferido, la composición de la invención comprende al menos dos agentes bactericidas que consisten en al menos 3-6% de peróxido de hidrógeno y al menos 2.2- 4.4% de ácido láctico y como agente quelante inhibidor de la bomba de eflujo al menos 12.5-25 mM de EDTA. En un aspecto preferido, la composición de la invención comprende al menos dos agentes bactericidas que consisten en 6% de peróxido de hidrógeno y 4.25% de ácido láctico y como agente quelante inhibidor de la bomba de eflujo 25 mM de EDTA. The third aspect of the present invention relates to a composition comprising at least two bactericidal agents consisting of hydrogen peroxide and lactic acid and EDTA as a chelating agent inhibitor of the efflux pump. In a preferred aspect, the composition of the invention comprises at least two bactericidal agents consisting of at least 3-6% hydrogen peroxide and at least 2.2-4.4% lactic acid and as a chelating agent inhibitor of the efflux pump minus 12.5-25 mM EDTA. In a preferred aspect, the composition of the invention comprises at least two bactericidal agents consisting of 6% hydrogen peroxide and 4.25% lactic acid and as a chelating agent inhibitor of the 25 mM EDTA efflux pump.
El cuarto aspecto de la invención hace referencia a un método ex vivo (fuera del cuerpo humano o animal) para la eliminación de biopelículas conformadas por al menos una bacteria, en cualquier utensilio o superficie, que comprende la aplicación de la composición de la invención arriba descrita. En un aspecto preferido, la composición de la invención se aplica a la biopelícula al menos durante 2 minutos, preferentemente entre 5 y 15 minutos. En un aspecto preferido, las bacterias que conforman la biopelícula, que serían eliminadas por la composición de la invención, se seleccionan del grupo que comprende: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 y Solmonello Enteritidis UJ3449. The fourth aspect of the invention refers to an ex vivo method (outside the human or animal body) for the elimination of biofilms formed by at least one bacterium, in any utensil or surface, comprising the application of the composition of the invention above described. In a preferred aspect, the composition of the invention is applied to the biofilm for at least 2 minutes, preferably between 5 and 15 minutes. In a preferred aspect, the bacteria that make up the biofilm, which would be eliminated by the composition of the invention, are selected from the group comprising: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 and Solmonello Enteritidis UJ3449.
El quinto aspecto de la presente invención hace referencia al uso ex vivo de la composición de la invención como desinfectante. En un aspecto preferido, la invención hace referencia al uso de la composición de la invención para la eliminación de biopelículas conformadas por al menos una bacteria. En un aspecto preferido la composición se aplica a la biopelícula al menos durante 2 minutos, preferentemente entre 5 y 15 minutos. En un aspecto preferido las bacterias que conforman la biopelícula
son: Staphylococcus oureus CECT 4468, Histeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 y Salmonella Enteritidis UJ3449. The fifth aspect of the present invention refers to the ex vivo use of the composition of the invention as a disinfectant. In a preferred aspect, the invention relates to the use of the composition of the invention for the elimination of biofilms formed by at least one bacterium. In a preferred aspect the composition is applied to the biofilm for at least 2 minutes, preferably between 5 and 15 minutes. In a preferred aspect the bacteria that make up the biofilm are: Staphylococcus oureus CECT 4468, Hysteria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 and Salmonella Enteritidis UJ3449.
El sexto aspecto de la presente invención hacer referencia a la composición de la invención para ser usada como medicamento, particularmente en el tratamiento de pacientes aquejados de infecciones bacterianas y/o de intoxicaciones causadas por metales pesados. En un aspecto preferido, la bacteria se selecciona de la lista que comprende: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 y Salmonella Enteritidis UJ3449. En otro aspecto preferido, el metal pesado es Cadmio. The sixth aspect of the present invention refers to the composition of the invention to be used as a medicine, particularly in the treatment of patients afflicted with bacterial infections and / or intoxications caused by heavy metals. In a preferred aspect, the bacterium is selected from the list comprising: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 and Salmonella Enteritidis UJ3449. In another preferred aspect, the heavy metal is Cadmium.
Es importante destacar que, en un aspecto particularmente preferido, la composición de la invención comprende unas concentraciones o cantidades precisamente establecidas con el objetivo de conseguir un efecto sinérgico entre sus componentes que dé lugar a la consecución de un triple efecto técnico: i) eliminación de la biopelícula en tiempos muy cortos de al menos 2 minutos, preferentemente entre 5-15 minutos, ii) sin generar toxicidad e iii) inhibiendo al mismo tiempo las bombas de eflujo responsables de la resistencia de las bacterias a agentes antimicrobianos. Las concentraciones o cantidades precisamente establecidas para conseguir dicho efecto sinérgico son: al menos 3-6% de peróxido de hidrógeno, al menos 2.2-4.4% de ácido láctico y al menos 12.5-25 mM de EDTA. En un aspecto preferido, la composición de la invención comprende 6% de peróxido de hidrógeno, 4.25% de ácido láctico y 25 mM de EDTA. It is important to note that, in a particularly preferred aspect, the composition of the invention comprises concentrations or quantities precisely established in order to achieve a synergistic effect between its components that leads to the achievement of a triple technical effect: i) elimination of the biofilm in very short times of at least 2 minutes, preferably between 5-15 minutes, ii) without generating toxicity and iii) inhibiting at the same time the efflux pumps responsible for the resistance of the bacteria to antimicrobial agents. The concentrations or amounts precisely established to achieve said synergistic effect are: at least 3-6% hydrogen peroxide, at least 2.2-4.4% lactic acid and at least 12.5-25 mM EDTA. In a preferred aspect, the composition of the invention comprises 6% hydrogen peroxide, 4.25% lactic acid and 25 mM EDTA.
A los efectos de la presente invención se definen los siguientes términos: For the purposes of the present invention, the following terms are defined:
• El término "que comprende" significa que incluye, pero no se limita a lo que sigue a la palabra "que comprende". Por lo tanto, el uso del término "que comprende" indica que los elementos enumerados son obligatorios u obligatorios, pero que otros elementos son opcionales y pueden o no estar presentes.
• Por "que consiste en" se entiende que incluye, y se limita a lo que sigue a la frase "que consiste en". Por lo tanto, la frase "que consiste en" indica que los elementos enumerados son obligatorios, y que no pueden estar presentes otros elementos. • The term "comprising" means that it includes, but is not limited to what follows the word "comprising". Therefore, the use of the term "comprising" indicates that the elements listed are mandatory or mandatory, but that other elements are optional and may or may not be present. • By "consisting of" is meant to include, and is limited to, what follows the phrase "consisting of". Therefore, the phrase "consisting of" indicates that the elements listed are mandatory, and that other elements can not be present.
• Por "desinfectante" se entiende un producto que permite eliminar, o permite evitar el establecimiento, de microorganismos en general y bacterias en particular. La utilización de un desinfectante permite limitar o, incluso, hacer desaparecer completamente la contaminación causada por los microorganismos. • "Disinfectant" means a product that eliminates or prevents the establishment of microorganisms in general and bacteria in particular. The use of a disinfectant allows to limit or even completely eliminate the contamination caused by microorganisms.
• Por "agente bactericida tóxico" se entiende cualquier agente bactericida que haya sido clasificado como tóxico en el estado de la técnica. Así, sería un agente capaz de provocar la muerte de las bacterias pero que al mismo tiempo exhibe una cierta toxicidad para el ser humano y los animales tras la exposición a dicho agente. Ejemplos de agentes bactericidas tóxicos son: fenoles, metales pesados, aldehidos y detergentes, particularmente ácido mandélico, ácido peracético, SDS, cloruro de cetilpiridinio, Sodio Cocoyl Sarcosinato, Lauril sarcosinato de sodio, Sodio dodecil difenil éter disulfonato. • "Bactericidal toxic agent" means any bactericidal agent that has been classified as toxic in the state of the art. Thus, it would be an agent capable of causing the death of bacteria but at the same time exhibiting a certain toxicity for humans and animals after exposure to said agent. Examples of toxic bactericidal agents are: phenols, heavy metals, aldehydes and detergents, particularly mandelic acid, peracetic acid, SDS, cetylpyridinium chloride, Sodium Cocoyl Sarcosinate, sodium lauryl sarcosinate, Sodium dodecyl diphenyl ether disulfonate.
Descripción de las figuras Description of the figures
Figura 1. Actividad antibacteriana de la composición de la invención en biopelículas mono y multiespecíficas (el cóctel de seis bacterias: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 y Salmonella Enteritidis UJ3449) durante 0 min (control), 5 minutos (T5), 10 minutos (TIO), 20 minutos (T20) y 30 minutos (T30) a temperatura ambiente según lo determinado por la determinación del recuento viable (Logio CFU/ml). En el eje Y se ilustra el Logio CFU/ml y el eje X las muestras. Figure 1. Antibacterial activity of the composition of the invention in mono and multispecific biofilms (the cocktail of six bacteria: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 and Salmonella Enteritidis UJ3449) for 0 min (control), 5 minutes (T5), 10 minutes (TIO), 20 minutes (T20) and 30 minutes (T30) at room temperature as determined by the determination of the viable count (Logio CFU / ml ). On the Y axis the Logio CFU / ml and the X axis samples are illustrated.
Figura 2. Visualización de la inactivación de la biopelícula multiespecífica utilizando el kit de viabilidad BacLight Uve / Dead (Invitrogen) después del tratamiento con la
composición de la invención (100% v / v) durante 0 minutos (A, Control), 2 minutos (B), 5 minutos (C) y 10 minutos (D) a temperatura ambiente y resuspensión de la biopelícula en PBS. Todas las imágenes se obtuvieron utilizando microscopio confocal LEICA TCS SP5 II (objetivo x63) y zoom de 2,5 (A y D), 1,5 (C) y 1 (B). Figure 2. Visualization of the inactivation of the multispecific biofilm using the BacLight Uve / Dead viability kit (Invitrogen) after the treatment with the composition of the invention (100% v / v) for 0 minutes (A, Control), 2 minutes (B), 5 minutes (C) and 10 minutes (D) at room temperature and resuspension of the biofilm in PBS. All images were obtained using LEICA TCS SP5 II confocal microscope (objective x63) and zoom of 2.5 (A and D), 1.5 (C) and 1 (B).
Figura 3. Fotos de microscopía confocal de biopelícula del cóctel de bacterias tratadas o no con el compuesto. Izquierda (control, células vivas), centro (tratamiento durante 5 minutos, gran cantidad de células muertas) y derecha (tratamiento durante 10 minutos, totalidad de células muertas). Figure 3. Photos of confocal microscopy of biofilm of the cocktail of bacteria treated or not with the compound. Left (control, live cells), center (treatment for 5 minutes, large amount of dead cells) and right (treatment for 10 minutes, all dead cells).
Figura 4. La composición de la invención adicionada a una concentración sub-inhibitoria (1/2 MIC) al cóctel de bacterias en TSB permitió la inhibición de la expresión de genes que codifican para las bombas de eflujo EfrAB (genes efrA y efrB) y NorE (el gen norE). C (Control). T (tratado con ½ MIC de la composición de la invención). Eje Y (Expresión relativa normalizada). Figure 4. The composition of the invention added to a sub-inhibitory concentration (1/2 MIC) to the cocktail of bacteria in TSB allowed the inhibition of the expression of genes coding for EffAB efflux pumps (efrA and efrB genes) and NorE (the norE gene). C (Control). T (treated with ½ MIC of the composition of the invention). Y axis (Normalized relative expression).
Descripción detallada de la invención Detailed description of the invention
MATERIAL Y MÉTODOS MATERIAL AND METHODS
Ejemplo 1. Cepas y condiciones de crecimiento bacteriano. Example 1. Strains and bacterial growth conditions.
En la presente invención se usaron Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 y Salmonella Enteritidis UJ3449. Las cepas se cultivaron en Tryptone Soya Broth (TSB) (Fluka, Madrid, España) a 37°C durante 24 horas. Los cultivos se mantuvieron en 20% de glicerol a -20°C y -80°C para almacenamiento a corto y largo plazo, respectivamente.
Ejemplo 2. Efecto de la composición de la invención sobre el crecimiento de células planctónicas. Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 and Salmonella Enteritidis UJ3449 were used in the present invention. Strains were grown in Tryptone Soya Broth (TSB) (Fluka, Madrid, Spain) at 37 ° C for 24 hours. The cultures were maintained in 20% glycerol at -20 ° C and -80 ° C for short and long term storage, respectively. Example 2. Effect of the composition of the invention on the growth of planktonic cells.
Para determinar la concentración mínima inhibitoria (MIC) y la concentración mínima de bactericida (MBC) de la composición de la invención (6% de H202, 4.25% de ácido láctico y 25 mM de EDTA), se utilizó el método de microdilución en caldo. Durante la noche, los cultivos bacterianos cultivados en caldo TSB a 37°C durante 24 horas se diluyeron 1/10 (v/v) en caldo TSB fresco y se agregaron 20 mI a cada pocilio de las placas de microtitulación de 96 pocilios. Posteriormente, se agregaron 180 mI de caldo TSB suplementado con la composición de la invención a diferentes concentraciones (0.25- 50%, v/v). Las placas se incubaron a 37°C en condiciones aerobias durante 24 horas y se evaluó el crecimiento bacteriano por la presencia de turbidez. Los pocilios que exhibieron la ausencia de turbidez se sometieron a determinación de recuento de viables (UFC/ml) mediante la siembra de las muestras (10 mI) en placas de agar triptona Soja (TSA). Posteriormente las placas se incubaron a 37°C durante 24 horas. La MIC se definió como la concentración más baja de la composición de la invención que inhibe el crecimiento visible y la MBC se definió como la concentración más baja de la composición de la invención que mata las bacterias (99%). Cada experimento fue hecho por triplicado. To determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the composition of the invention (6% H 2 0 2 , 4.25% lactic acid and 25 mM EDTA), the method of microdilution in broth. Overnight, bacterial cultures grown in TSB broth at 37 ° C for 24 hours were diluted 1/10 (v / v) in fresh TSB broth and 20 ml were added to each well of the 96-well microtiter plates. Subsequently, 180 ml of TSB broth supplemented with the composition of the invention were added at different concentrations (0.25-50%, v / v). The plates were incubated at 37 ° C under aerobic conditions for 24 hours and bacterial growth was evaluated by the presence of turbidity. The wells that exhibited the absence of turbidity were subjected to viable count determination (CFU / ml) by sowing the samples (10 ml) on Tryptone Soy agar plates (TSA). Subsequently the plates were incubated at 37 ° C for 24 hours. MIC was defined as the lowest concentration of the composition of the invention that inhibits visible growth and MBC was defined as the lowest concentration of the composition of the invention killing the bacteria (99%). Each experiment was done in triplicate.
Ejemplo 3. Determinación de la actividad anti-biopelícula. Example 3. Determination of the anti-biofilm activity.
Las propiedades anti-adherencia de la composición de la invención a diferentes cepas bacterianas (S. aureus CECT 4468, L. monocytogenes CECT 4032, E. faecalis S-47, B. cereus CECT 5148, E. coli CCUG 47553 y S. Enteritidis UJ3449) y el cóctel de todas las cepas se ensayaron en placas de microtitulación. Durante la noche, los cultivos bacterianos cultivados en caldo TSB a 37°C durante 24 horas se diluyeron 1/10 (v/v) en caldo TSB fresco y se agregaron 20 mI a cada pocilio de las placas de microtitulación de 96 pocilios. Posteriormente, los pocilios se completaron con 180 mI de caldo TSB suplementado con la composición de la invención a concentraciones sub-MIC (1/2 MIC de la composición de la invención para cada cepa hasta el 20% de la composición de la invención, v/v). Los
controles sin la composición de la invención consistieron en 180 pl de caldo TSB. Las placas se incubaron a 37°C en condiciones aerobias durante 24 horas y los pocilios se lavaron con tampón fosfato salino (PBS). La actividad anti-biopelícula de la composición de la invención se determinó tiñendo los pocilios lavados con 100 mI de cristal violeta al 1% (p/v) que se incubaron a temperatura ambiente durante 15 minutos. Posteriormente, se determinó la absorbancia a 590 nm usando un lector de microplacas (lector de absorbancias de microplacas ¡Mark, instrumento Bio-Rad). El porcentaje de inhibición de la formación de biopelículas se determinó usando la siguiente fórmula como se describe por Zmantar et al. (2017): The anti-adhesion properties of the composition of the invention to different bacterial strains (S. aureus CECT 4468, L. monocytogenes CECT 4032, E. faecalis S-47, B. cereus CECT 5148, E. coli CCUG 47553 and S. Enteritidis UJ3449) and the cocktail of all strains were tested in microtiter plates. Overnight, bacterial cultures grown in TSB broth at 37 ° C for 24 hours were diluted 1/10 (v / v) in fresh TSB broth and 20 ml were added to each well of the 96-well microtiter plates. Subsequently, the wells were completed with 180 ml of TSB broth supplemented with the composition of the invention at sub-MIC concentrations (1/2 MIC of the composition of the invention for each strain up to 20% of the composition of the invention, v / v). The controls without the composition of the invention consisted of 180 pl of TSB broth. The plates were incubated at 37 ° C under aerobic conditions for 24 hours and the wells were washed with phosphate buffered saline (PBS). The anti-biofilm activity of the composition of the invention was determined by staining the wells washed with 100 ml of 1% (w / v) violet crystal which were incubated at room temperature for 15 minutes. Subsequently, the absorbance at 590 nm was determined using a microplate reader (microplate absorbance reader Mark, Bio-Rad instrument). The percent inhibition of biofilm formation was determined using the following formula as described by Zmantar et al. (2017):
DO: densidad óptica. j ¡ DO Control - DO Muestra | j DO: optical density. j DO Control - DO Sample | j
j i X 100 j i X 100
j | DO Control | j j | DO Control | j
Ejemplo 4. Efecto antimicrobiano de la composición de la invención sobre la biopelícula preformada. Example 4. Antimicrobial effect of the composition of the invention on the preformed biofilm.
Se usó un inoculo con el 2% de cada bacteria y el cóctel de todas las cepas en TSB para la preparación de la biopelícula que se cultivó en placas de microtitulación de 96 pocilios durante 24 horas 37°C. Después de la incubación, el caldo de cultivo que contenía bacterias no adheridas se retiró y los pocilios se lavaron con PBS estéril. Las biopelículas se trataron con la composición de la invención durante diferentes períodos de tiempo (5, 10, 15, 20 y 30 minutos) a temperatura ambiente. Después de los tratamientos, se retiró la composición de la invención y los pocilios se incubaron con 200 mI de caldo neutralizante D / E (Difeo, Barcelona) durante 5 minutos a temperatura ambiente y luego se lavaron con 200 mI de PBS. Las biopelículas se resuspendieron en 200 mI de PBS y luego se sembraron en TSA. Las placas se incubaron a 37°C durante 24 horas para la determinación del número total de UFC/ml.
Ejemplo 5. Evaluación microscópica del efecto de la composición de la invención en la biopelícula. An inoculum with 2% of each bacterium and the cocktail of all strains in TSB was used for the preparation of the biofilm which was cultured in 96-well microtiter plates for 24 hours at 37 ° C. After incubation, the culture broth containing unattached bacteria was removed and the wells were washed with sterile PBS. The biofilms were treated with the composition of the invention for different periods of time (5, 10, 15, 20 and 30 minutes) at room temperature. After the treatments, the composition of the invention was removed and the wells were incubated with 200 ml of neutralizing broth D / E (Difeo, Barcelona) for 5 minutes at room temperature and then washed with 200 ml of PBS. The biofilms were resuspended in 200 ml of PBS and then seeded in TSA. The plates were incubated at 37 ° C for 24 hours for the determination of the total number of CFU / ml. Example 5. Microscopic evaluation of the effect of the composition of the invention on the biofilm.
La obtención de imágenes de la biopelícula tratada con la composición de la invención se realizó utilizando LIVE/DEAD BacLight™ (Thermo Fisher Scientific, Waltham, MA, EE. UU) y un microscopio de escaneo láser confocal (LEICA TCS-SP5 II, Mannheim, Alemania) equipado con Plan-Apochromat 63x / 1.4 objetivo. Después de cultivar la biopelícula en una placa de microtitulación (200 pl) como se describió anteriormente, algunos pocilios no se trataron con la composición de la invención (control) y los otros se sometieron a tratamiento con la composición de la invención durante 5 y 10 minutos a temperatura ambiente, se lavaron con PBS estéril y se resuspendieron en 50 mI de PBS. Posteriormente, 20 mI de las suspensiones (control y tratamiento con la composición de la invención) fueron completadas con 0,5 mI de tinción LIVE/DEAD y posteriormente se colocaron en un portaobjetos de vidrio y se obtuvieron imágenes usando un microscopio de escaneo láser confocal. The imaging of the biofilm treated with the composition of the invention was performed using LIVE / DEAD BacLight ™ (Thermo Fisher Scientific, Waltham, MA, USA) and a confocal laser scanning microscope (LEICA TCS-SP5 II, Mannheim , Germany) equipped with Plan-Apochromat 63x / 1.4 objective. After culturing the biofilm in a microtiter plate (200 pl) as described above, some wells were not treated with the composition of the invention (control) and the others were treated with the composition of the invention for 5 and 10 minutes. minutes at room temperature, washed with sterile PBS and resuspended in 50 ml of PBS. Subsequently, 20 ml of the suspensions (control and treatment with the composition of the invention) were completed with 0.5 ml of LIVE / DEAD stain and subsequently placed on a glass slide and images were obtained using a confocal laser scanning microscope .
Ejemplo 6. Efecto de la concentración sub-inhibitoria de la composición de la invención sobre la resistencia a la biopelícula. Example 6. Effect of the sub-inhibitory concentration of the composition of the invention on the resistance to the biofilm.
Se añadió (o no) 1/2 MIC de la composición de la invención en caldo TSB (2 mi) al cóctel de las seis cepas bacterianas (2%) y posteriormente se incubaron durante 18 horas a 37°C en tubos y también en placas de microtitulación de 12 pocilios para formación de biopelículas. La extracción de ARN se realizó a partir de cultivos bacterianos planctónicos y también de biopelículas multiespecie utilizando el kit de ARN Direct-zol ™ (Zymo Research, EE. UU) de acuerdo con las instrucciones del fabricante. (1/2) MIC of the composition of the invention in TSB broth (2 ml) was added (or not) to the cocktail of the six bacterial strains (2%) and subsequently incubated for 18 hours at 37 ° C in tubes and also in 12-well microtiter plates for biofilm formation. RNA extraction was performed from planktonic bacterial cultures and also from multispecies biofilms using Direct-zol ™ RNA kit (Zymo Research, USA) according to the manufacturer's instructions.
Ejemplo 7. Análisis estadístico.
Todos los análisis se realizaron por triplicado. Los análisis estadísticos se realizaron utilizando el programa Excel 2007 (Microsoft Corporation, Redmond, Washington, EE. UU). Para determinar los promedios y las desviaciones estándar. La evaluación estadística de la inhibición del desarrollo de biopelículas se realizó por análisis de varianza (ANOVA) usando el software Statgraphics Centurión XVI (Statpoint Technologie, Warrenton, Virginia, EE. UU). El mismo software se usó para realizar las pruebas de Shapiro-Wilk y Levene para verificar la normalidad de los datos y realizar el contraste múltiple de Tukey bilateral para determinar las diferencias por pares entre las cepas, donde el nivel de significancia se estableció en el valor P de <0.05. Example 7. Statistical analysis. All analyzes were performed in triplicate. The statistical analyzes were performed using the Excel 2007 program (Microsoft Corporation, Redmond, Washington, USA). To determine averages and standard deviations. The statistical evaluation of the inhibition of biofilm development was performed by analysis of variance (ANOVA) using the Statgraphics Centurion XVI software (Statpoint Technologie, Warrenton, Virginia, USA). The same software was used to perform the Shapiro-Wilk and Levene tests to verify the normality of the data and to perform the multiple Tukey bilateral contrast to determine the differences by pairs between the strains, where the level of significance was established in the value P of <0.05.
RESULTADOS RESULTS
Ejemplo 8. Actividad antimicrobiana de la composición de la invención en cultivos planctónicos. Example 8. Antimicrobial activity of the composition of the invention in planktonic cultures.
La Tabla 1 muestra el efecto antimicrobiano que ejerce la composición de la invención sobre el crecimiento de las células planctónicas de cada bacteria y también sobre el cóctel de todas las bacterias ensayadas. La Tabla 1 muestra la determinación de la Concentración Inhibidora Mínima (MIC) y Concentración Bactericida Mínima (MBC) de la composición de la invención. Table 1 shows the antimicrobial effect exerted by the composition of the invention on the growth of the planktonic cells of each bacterium and also on the cocktail of all the bacteria tested. Table 1 shows the determination of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the composition of the invention.
Tabla 1 Table 1
Cepas MIC de HLE (%, v/v) MBC de HLE (%, v/v) MIC strains of HLE (%, v / v) MBC of HLE (%, v / v)
E. faecalis S-47 0.25 0.25 E. faecalis S-47 0.25 0.25
L. monocytogenes CECT 4032 0.15 0.2 L. monocytogenes CECT 4032 0.15 0.2
S. aureus CECT 4468 0.4 0.4 S. aureus CECT 4468 0.4 0.4
B. cereus CECT 5148 0.3 0.5 B. cereus CECT 5148 0.3 0.5
E. coli CCUG 47553 0.3 0.5 E. coli CCUG 47553 0.3 0.5
S. Enteritidis ETJ3449 0.3 0.4 S. Enteritidis ETJ3449 0.3 0.4
Cocktail 0.3 0.5
Se detectó una alta susceptibilidad de las cepas bacterianas planctónicas ya que exhibían MIC muy bajas que variaban de 0.15% a 0.4% de la composición de la invención (v/v) siendo L. monocytogenes CECT 4032 y E. faecalis S-47 las cepas más susceptibles. Sin embargo, S. aureus CECT 4468 fue menos susceptible que otras especies y también el cóctel. En cuanto a los valores de MBC, fueron muy similares, oscilando entre 0.2% y 0.5% de la composición de la invención (v/v). En este sentido, la composición de la invención fue efectiva contra bacterias Gram-positivas y Gram-negativas y además frente al cóctel hecho con las seis bacterias ya que también fue eliminado a la vista de la baja concentración de la composición de la invención (0.5%). Cocktail 0.3 0.5 A high susceptibility of planktonic bacterial strains was detected since they exhibited very low MICs ranging from 0.15% to 0.4% of the composition of the invention (v / v) with L. monocytogenes CECT 4032 and E. faecalis S-47 strains being more susceptible. However, S. aureus CECT 4468 was less susceptible than other species and also the cocktail. As for the MBC values, they were very similar, ranging between 0.2% and 0.5% of the composition of the invention (v / v). In this sense, the composition of the invention was effective against Gram-positive and Gram-negative bacteria and also against the cocktail made with the six bacteria since it was also eliminated in view of the low concentration of the composition of the invention (0.5 %).
Ejemplo 9. Inhibición de la formación de biopelículas por la composición de la invención. Example 9. Inhibition of biofilm formation by the composition of the invention.
Se logró una fuerte inhibición del desarrollo de biopelículas usando la composición de la invención para cada cepa y para el cóctel bacteriano. Los resultados obtenidos mostraron que se logró el 80-91% de la inhibición de la biopelícula en desarrollo para cepas individuales y también el cóctel (Tabla 2). Además, el uso de 1/2 MIC de la composición de la invención permitió una inhibición del 33-50% del desarrollo de la biopelícula (Tabla 2). Estos resultados indicaron que la composición de la invención inhibió la adherencia bacteriana al poliestireno (Tabla 2). Strong inhibition of biofilm development was achieved using the composition of the invention for each strain and for the bacterial cocktail. The results obtained showed that 80-91% of the inhibition of the biofilm in development was achieved for individual strains and also the cocktail (Table 2). In addition, the use of 1/2 MIC of the composition of the invention allowed an inhibition of 33-50% of the biofilm development (Table 2). These results indicated that the composition of the invention inhibited bacterial adhesion to polystyrene (Table 2).
Tabla 2 Table 2
Cepas Inhibición del desarrollo de las biopelículas Strains Inhibition of the development of biofilms
L. monocytogenes CECT 4032 48 ± 0.05b 85 ± 0.06a L. monocytogenes CECT 4032 48 ± 0.05 b 85 ± 0.06 a
S. aureus CECT 4468 50 ± 0.04b 91 ± 0.02a S. aureus CECT 4468 50 ± 0.04 b 91 ± 0.02 a
B. cereus CECT 5148 33 ± 0.01a 80 ± 0.02b B. cereus CECT 5148 33 ± 0.01 to 80 ± 0.02 b
E. coli CCUG 47553 46 ± 0.02b 90 ± 0.01a
S. Enteritidis UJ3449 49 ± 0.04b 85 ± 0.30c E. coli CCUG 47553 46 ± 0.02 b 90 ± 0.01 a S. Enteritidis UJ3449 49 ± 0.04 b 85 ± 0.30 c
_ Cocktail _ 47 ± 0.02b _ 83 ± 0.0lc _ _ Cocktail _ 47 ± 0.02 b _ 83 ± 0.0l c _
±DS, desviación estándar de tres experimentos independientes. ± DS, standard deviation of three independent experiments.
*Cada letra minúscula diferente representa diferencias significativas según el HSD de Tukey entre las cepas (p <0.05). * Each different lowercase letter represents significant differences according to the Tukey HSD between the strains (p <0.05).
Ejemplo 10. Evaluación del efecto antimicrobiano de la composición de la invención en biopelículas. Example 10. Evaluation of the antimicrobial effect of the composition of the invention in biofilms.
Las biopelículas preformadas de diferentes cepas bacterianas en placas de microtitulación se sometieron al efecto antimicrobiano de la composición de la invención durante varios tiempos de contacto (5, 10, 15, 20 y 30 minutos). Los resultados obtenidos mostraron el efecto bactericida total de la composición de la invención contra todas las cepas bacterianas y también el cóctel después de todos los tiempos de contacto que varían de 5 a 30 minutos (ver Figura 1). Además, la microscopía confocal reveló que la composición de la invención tenía un fuerte efecto sobre la biopelícula multiespecie ya que no se detectaron células viables de las biopelículas tratadas con la composición de la invención (5 y 10 minutos) dentro de la matriz de la biopelícula (ver Figura 2 B-D), en comparación con controles no tratados con la composición de la invención (ver Figura 2 A). Sin embargo, se visualizaron células muertas (ver Figura 2 C y D). The preformed biofilms of different bacterial strains in microtiter plates were subjected to the antimicrobial effect of the composition of the invention during several contact times (5, 10, 15, 20 and 30 minutes). The results obtained showed the total bactericidal effect of the composition of the invention against all bacterial strains and also the cocktail after all contact times ranging from 5 to 30 minutes (see Figure 1). In addition, confocal microscopy revealed that the composition of the invention had a strong effect on the multispecies biofilm since no viable cells of the biofilms treated with the composition of the invention (5 and 10 minutes) were detected within the biofilm matrix. (see Figure 2 BD), compared to controls not treated with the composition of the invention (see Figure 2 A). However, dead cells were visualized (see Figure 2 C and D).
Ejemplo 11. La composición de la invención inhibe las bombas de eflujo. Example 11. The composition of the invention inhibits the efflux pumps.
La composición de la invención es capaz de inhibir las bombas de eflujo que actúan como mecanismos de resistencia inespecífica en las bacterias (particularmente bombas de eflujo EfrAB y NorE). Esto se demuestra en la presente invención mediante análisis de la expresión de dichos genes en el cóctel de las bacterias estudiadas en ausencia y presencia de bajas concentraciones del compuesto (1/2 MIC). La composición de la invención adicionada a una concentración sub-inhibitoria (1/2 MIC) al cóctel de bacterias en TSB permitió la inhibición de la expresión de genes que codifican para las bombas de eflujo EfrAB (genes efrA y efrB) y NorE (el gen norE) tal y como se muestra en la Figura 4. Esta
inhibición de las bombas de eflujo reduce la diseminación de patógenos resistentes a los diferentes antimicrobianos. Así, se evita el desarrollo de biopelículas con mayor resistencia a los antimicrobianos. En la Figura 4 se ve claramente que la aplicación de la composición de la invención a una concentración sub-inhibitoria del crecimiento (1/2 MIC) tiene una baja expresión de los genes codificadores de las bombas de eflujo EfrAB y NorE, las cuales juegan un papel importante en la resistencia intrínseca e inespecífica a los diferentes agentes antimicrobianos (antibióticos y biocidas). Por lo tanto, por un lado, gracias a la acción inhibitoria de las bombas de eflujo, la composición de la invención permite potenciar la acción antimicrobiana de los dos agentes bactericidas (ácido láctico y peróxido de hidrógeno) al bloquear uno de los mecanismos de defensa bacteriana, permitiendo así restaurar la actividad antimicrobiana de diversos compuestos antimicrobianos que han perdido su actividad debido a la presencia de bombas de eflujo. Por otra parte, el uso de la composición de la invención, al bloquear o inhibir las bombas de eflujo, reducirá la emergencia existente relativa a la existencia de bacterias multirresistentes a los antimicrobianos. Actualmente, las bombas de eflujo representan unas dianas muy atractivas para reducir la diseminación de la resistencia a los antimicrobianos (antibióticos y biocidas). Particularmente, el mecanismo mediante el cual se cree que el EDTA promueve la inhibición de la bomba de eflujo es mediante el secuestro de los iones de calcio, así, no puede producirse la hidrólisis del ATP y no puede ponerse en funcionamiento las bombas de exporte de la familia de los transportadores ABC, como es el caso de la bomba de exporte EfrAB.
The composition of the invention is capable of inhibiting efflux pumps that act as non-specific resistance mechanisms in bacteria (particularly EfrAB and NorE efflux pumps). This is demonstrated in the present invention by analysis of the expression of said genes in the cocktail of bacteria studied in the absence and presence of low concentrations of the compound (1/2 MIC). The composition of the invention added to a sub-inhibitory concentration (1/2 MIC) to the bacterial cocktail in TSB allowed the inhibition of the expression of genes coding for efflow pumps EfrAB (efrA and efrB genes) and NorE (the norE gene) as shown in Figure 4. This Inhibition of efflux pumps reduces the spread of pathogens resistant to different antimicrobials. Thus, the development of biofilms with greater resistance to antimicrobials is avoided. In Figure 4 it is clearly seen that the application of the composition of the invention to a sub-inhibitory concentration of growth (1/2 MIC) has a low expression of the coding genes of the EffAB and NorE efflux pumps, which play an important role in the intrinsic and non-specific resistance to the different antimicrobial agents (antibiotics and biocides). Therefore, on the one hand, thanks to the inhibitory action of the efflux pumps, the composition of the invention allows to potentiate the antimicrobial action of the two bactericidal agents (lactic acid and hydrogen peroxide) by blocking one of the defense mechanisms bacterial, thus allowing to restore the antimicrobial activity of various antimicrobial compounds that have lost their activity due to the presence of efflux pumps. On the other hand, the use of the composition of the invention, by blocking or inhibiting the efflux pumps, will reduce the existing emergency relative to the existence of multiresistant bacteria to the antimicrobials. Currently, efflux pumps represent very attractive targets to reduce the spread of resistance to antimicrobials (antibiotics and biocides). Particularly, the mechanism by which EDTA is believed to promote the inhibition of the efflux pump is by sequestering the calcium ions, thus, the hydrolysis of the ATP can not occur and the export pumps can not be put into operation. the family of ABC transporters, as is the case with the EfrAB export pump.
Claims
1. Composición que comprende peróxido de hidrógeno, ácido láctico y EDTA, caracterizada por no comprender agentes bactericidas tóxicos. 1. Composition comprising hydrogen peroxide, lactic acid and EDTA, characterized by not comprising toxic bactericidal agents.
2. Composición, según la reivindicación 1, caracterizada porque el agente bactericida tóxico se selecciona de la lista que comprende: fenoles, metales pesados, aldehidos y detergentes. Composition according to claim 1, characterized in that the toxic bactericidal agent is selected from the list comprising: phenols, heavy metals, aldehydes and detergents.
3. Composición, según cualquiera de las reivindicaciones anteriores, caracterizada porque el agente bactericida tóxico se selecciona de la lista que comprende: ácido mandélico, ácido peracético, SDS, cloruro de cetilpiridinio, Sodio Cocoyl Sarcosinato, Lauril sarcosinato de sodio y/o Sodio dodecil difenil éter disulfonato. Composition according to any one of the preceding claims, characterized in that the toxic bactericidal agent is selected from the list comprising: mandelic acid, peracetic acid, SDS, cetylpyridinium chloride, Sodium Cocoyl Sarcosinate, sodium lauryl sarcosinate and / or Sodium dodecyl diphenyl ether disulfonate.
4. Composición, según cualquiera de las reivindicaciones anteriores, que comprende: a. Al menos 3-6% de peróxido de hidrógeno. 4. Composition, according to any of the preceding claims, comprising: a. At least 3-6% hydrogen peroxide.
b. Al menos 2.2-4.4% de ácido láctico. b. At least 2.2-4.4% lactic acid.
c. 12.5-25 mM de EDTA. c. 12.5-25 mM EDTA.
5. Composición, según cualquiera de las reivindicaciones anteriores, que comprende: a. 6% de peróxido de hidrógeno. 5. Composition according to any of the preceding claims, comprising: a. 6% hydrogen peroxide.
b. 4.25% de ácido láctico. b. 4.25% lactic acid.
c. 25 mM de EDTA. c. 25 mM EDTA.
6. Método para la eliminación de biopelículas conformadas por al menos una bacteria, presentes en cualquier superficie o utensilio, que comprende la aplicación de la composición de la reivindicaciones 1 a 5. Method for the elimination of biofilms formed by at least one bacterium, present in any surface or utensil, which comprises the application of the composition of claims 1 to 5.
7. Método, según la reivindicación 6, donde la composición se aplica a la biopelícula al menos durante 2 minutos, preferentemente durante 5-15 minutos.
The method according to claim 6, wherein the composition is applied to the biofilm for at least 2 minutes, preferably for 5-15 minutes.
8. Método, según las reivindicaciones 6 o 7, donde las bacterias que conforman la biopelícula son: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 y Solmonello Enteritidis UJ3449. 8. Method according to claim 6 or 7, wherein the bacteria that make up the biofilm are: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 and Solmonello Enteritidis UJ3449.
9. Uso de la composición de las reivindicaciones 1 a 5 como desinfectante de cualquier tipo de utensilio o superficie. 9. Use of the composition of claims 1 to 5 as a disinfectant of any type of utensil or surface.
10. Uso, según la reivindicación 9, para la eliminación de biopelículas conformadas por al menos una bacteria. 10. Use, according to claim 9, for the elimination of biofilms formed by at least one bacterium.
11. Uso, según las reivindicaciones 9 o 10, donde la composición se aplica a la biopelícula al menos durante 2 minutos, preferentemente durante 5-15 minutos. 11. Use according to claims 9 or 10, wherein the composition is applied to the biofilm for at least 2 minutes, preferably for 5-15 minutes.
12. Uso, según las reivindicaciones 9 a 11, donde las bacterias que conforman la biopelícula son: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 y Salmonella Enteritidis UJ3449. 12. Use according to claims 9 to 11, wherein the bacteria that make up the biofilm are: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 and Salmonella Enteritidis UJ3449.
13. Composición, según cualquiera de las reivindicaciones 1 a 5, para ser usada en el tratamiento de pacientes aquejados de infecciones bacterianas y/o de intoxicaciones causadas por metales pesados. 13. Composition according to any of claims 1 to 5, for use in the treatment of patients suffering from bacterial infections and / or intoxications caused by heavy metals.
14. Composición para ser usada, según la reivindicación 13, donde la bacteria se seleccionada de la lista que comprende: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 y Salmonella Enteritidis UJ3449. 14. Composition for use according to claim 13, wherein the bacterium is selected from the list comprising: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 and Salmonella Enteritidis UJ3449.
15. Composición para ser usada, según la reivindicación 13, donde el metal pesado es Cadmio.
15. Composition to be used, according to claim 13, wherein the heavy metal is Cadmium.
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| ES201731462A ES2717798B2 (en) | 2017-12-22 | 2017-12-22 | Disinfectant composition |
| ESP201731462 | 2017-12-22 |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993002973A1 (en) * | 1991-08-05 | 1993-02-18 | Trawöger, Werner | Anti-fouling agent for wet surfaces |
| US5731275A (en) * | 1994-04-05 | 1998-03-24 | Universite De Montreal | Synergistic detergent and disinfectant combinations for decontaminating biofilm-coated surfaces |
| US20020016278A1 (en) * | 1998-11-06 | 2002-02-07 | Jean Barbeau | Bactericidal and non-bactericidal solutions for removing biofilms. |
| US20050153031A1 (en) * | 2004-01-09 | 2005-07-14 | Ecolab Inc. | Methods for washing carcasses, meat, or meat products with medium chain peroxycarboxylic acid compositions |
| US20090312279A1 (en) * | 2005-12-23 | 2009-12-17 | Sterilex Technologies, Llc | Antimicrobial compositions |
-
2017
- 2017-12-22 ES ES201731462A patent/ES2717798B2/en active Active
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2018
- 2018-12-05 WO PCT/ES2018/070785 patent/WO2019122470A1/en active Application Filing
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993002973A1 (en) * | 1991-08-05 | 1993-02-18 | Trawöger, Werner | Anti-fouling agent for wet surfaces |
| US5731275A (en) * | 1994-04-05 | 1998-03-24 | Universite De Montreal | Synergistic detergent and disinfectant combinations for decontaminating biofilm-coated surfaces |
| US20020016278A1 (en) * | 1998-11-06 | 2002-02-07 | Jean Barbeau | Bactericidal and non-bactericidal solutions for removing biofilms. |
| US20050153031A1 (en) * | 2004-01-09 | 2005-07-14 | Ecolab Inc. | Methods for washing carcasses, meat, or meat products with medium chain peroxycarboxylic acid compositions |
| US20090312279A1 (en) * | 2005-12-23 | 2009-12-17 | Sterilex Technologies, Llc | Antimicrobial compositions |
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| ES2717798B2 (en) | 2020-09-22 |
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