WO2014020224A1

WO2014020224A1 - Method for preventing and/or controlling bacterial infections

Info

Publication number: WO2014020224A1
Application number: PCT/ES2013/070570
Authority: WO
Inventors: María Jose SOTO MISFFUT; Joaquina NOGALES DÍAZ; José OLIVARES PASCUAL; Juan SAN JUÁN PINILLA
Original assignee: Consejo Superior De Investigaciones Científicas (Csic)
Priority date: 2012-08-01
Filing date: 2013-07-31
Publication date: 2014-02-06
Also published as: ES2445491A1; ES2445491B1

Abstract

The invention relates to a method for preventing and/or controlling bacterial infections in a plant and/or culture, and to products (raw food, washing solutions, material for use in hospitals) and facilities that are susceptible to contamination by bacteria, by means of the application of long-chain aliphatic methyl ketones, essentially 2-tridecanone but also 2-undecanone, 2-dodecanone, and 2-pentadecanone, in non-toxic forms and concentrations of a micromolar order such that they enable the prevention of said infection without producing a bactericide effect (destruction of bacteria). The invention also relates to said methyl ketones for use in medicine, by means of the administration of said compound to an animal or a human being.

Description

METHOD TO PREVENT AND / OR CONTROL BACTERIAL INFECTIONS

Field of the Invention

The present invention is encompassed in multiple areas of application, such as Chemistry and Pharmacy, specifically in the field of Agriculture, using the product in question as a phytosatinary intended to reduce diseases in plants caused by bacteria, and prevent foodborne diseases caused for the ingestion of fresh vegetable products contaminated with pathogenic bacteria. The invention also has application in the field of Medicine and Health, to prevent and treat bacterial infections of clinical and veterinary importance in animals and people, as well as in facilities susceptible to contamination by bacteria, such as the food industry.

State of the art

Control of plant diseases caused by bacteria

(bacteriosis)

The most frequent and usual way to protect plants against the attack of pathogens has been based on the massive use of pesticides. The damage caused by these chemical compounds to the environment as well as their potential risks to animal and human health, require the search for alternative solutions for crop protection.

Phytopathogenic bacteria are the causative agent of numerous diseases in plants. These diseases are normally difficult to control, so the measures are aimed primarily at prevention. Among the chemical agents used to control this type of infections are those based on copper such as Bordeaux broth, copper oxychloride or cupric hydroxide. However, these compounds do not give satisfactory results when the Environmental conditions are optimal for the development and spread of the pathogen. In addition, the appearance of bacteria resistant to these compounds is frequent. Another way to fight bacterial infections in plants is to use antibiotics such as streptomycin and oxytetracycline. The drawback of these treatments is that they can not be used on plants intended for human and animal consumption as well as the frequent occurrence of resistance (Agrios 2005. Plant Pathology, 5th edition, Academic Press).

An alternative to these treatments that is being investigated in recent years is the use of antimicrobial peptides that are well produced by living, very synthetic organisms (Marcos et al. 2008. Annu. Rev. Phytopathol. 46: 273-301). The main drawbacks of its use in the control of bacteriosis are in its limited efficacy in vivo since they are degraded by proteases of plant tissues, or in their high economic cost in the case of synthetics.

Prevention of food diseases caused by the ingestion of fresh plant products contaminated with pathogenic bacteria In recent years there have been numerous evidences that indicate that fruits, herbs, vegetables, and especially green leafy vegetables, are authentic reservoirs of enteropathogenic bacteria such as Enteric salmonella or Escherichia coli (Berger et al. 2010. Environ. Microbiol. 12: 2385-2397). The origin of the infection can be diverse, with irrigation water being the main route of contamination. Since this type of food is usually consumed raw or with very little processing, the presence in them of this type of pathogens constitutes a risk to human health. We have had a close example of this problem in the food crisis that took place in Germany in 2011 caused by an aggressive strain of E. coli that caused the death of several people and the poisoning of several hundreds. He The origin of this poisoning was initially attributed to cucumber items of Spanish origin, which caused an alarm that, although later denied, caused serious losses in the Spanish fruit and vegetable sector.

The domestic prevention of this type of infections is mainly based on washing thoroughly with water and peeling whenever possible. The use of lemon juice and / or vinegar before consumption seems to be effective in reducing the number of viable cells of some of these pathogens. Producers and distributors usually include practices to eliminate this type of pollutants based on post-harvest disinfectant treatments with chlorine (20-200 μg / ml), ozone or a combination of both, or ionizing radiation. Another alternative suggested to reduce the risk of contamination consists in the development of agricultural practices that increase bacterial populations in the field that are good competitors against potential pathogens (reviewed in Berger et al. 2010. Environ. Microbiol. 12: 2385-2397 ).

Control of infectious diseases in animals and humans caused by bacteria The most common and effective procedure in the treatment of bacterial infections in animals and humans is the administration of antibiotics. Antibiotics act causing the death of the bacteria or preventing their multiplication. The sometimes indiscriminate use of these drugs is leading to the emergence of bacteria resistant to the action of the most common antibiotics (super-bugs). Diseases caused by resistant bacteria are usually very serious because they are difficult to treat. In the best case, they lead to an increase in the cost of treatment, since it is necessary to make use of new generation antibiotic products against which resistance has not yet appeared. In the case of resistance to beta-lactam antibiotics, it is giving good The combined treatment of beta-lactam with an inhibitor of beta-lactamases, enzymes produced by the bacteria that inactivate the antibiotic. Phage therapies or phage products also appear to be effective in treating or even preventing bacterial infections (Hermoso et al. 2007. Curr. Opin. Microbiol. 10: 461-472).

2-Tridecanone 2-Tridecanone (2-TDC, methyl undecyl ketone) is a long-chain aliphatic methyl ketone (Fig. 1). Aliphatic methyl ketones are volatile natural compounds that have been identified in microorganisms, plants, insects and animals (Forney and Markovetz. 1971. The biology of methyl ketones. J. Lipid Res. 12: 383-395).

2-TDC is especially abundant in leaves of wild tomato Solanum habrochaites subsp. glabratum in which it can accumulate in concentrations up to 70 times higher (9.4 μg of 2-TDC per mg fresh leaf weight) than those found in the cultivable tomato variety (Williams et al. 1980. 2-Tridecanone: A Naturally Occurring Insecticide from the Wild Tomato Lycopersicon hirsutum F. glabratum. Science 207: 888-889; Anthonius. 2001. Production and quantification of methyl ketones in wild tomato accessions. J. Environ. Sel Health B 36: 835-848). However, 2-TDC does not appear to be tomato exclusive and has been identified as a relatively abundant component in the roots of Glossocalyx staudtii, in seeds of Pilocarpus microphyllus and in leaves of Zanthoxylum armatum (Agnaniet et al. 2009. Chemical composition and antimicrobial activity of the essential oils of leaves, roots and bark of Glossocalyx staudtii. Nat. Prod. Commun. 4: 1127-1132; Braga et al. 2007. Insecticidal activity of 2-tridecanone against the cowpea weevil Callosobruchus maculatus (Coleoptera: Bruchidae ) An.Acad.Bras. Cieñe. 79: 35-39; Bisht and Chanotiya. 2011. 2- undecanone rich leaf essential oil from Zanthoxylum armatum. Nat Prod. Commun. 6: 111-114). 2-TDC has also been identified in animals: as a major component in the secretion of the interdigital glands of the Odocoileus hemionus columbianus deer, where it could participate in semi-chemical communication (Wood et al. 1995. Volatile ketones from interdigital glands of black-tailed deer, Odocoileus hemionus columbianus. J. Chem Ecol. 21: 1401-1408), and in mandibular glands of worker bees where it seems to act as a pheromone that induces aggressive responses in the insect (Schorkopf et al. 2009. Mandibular gland secretions of meliponine worker bees: further evidence for their role in interspecific and intraspecific defense and aggression and against their role in food source signalling. J. Exp. Biol. 212: 1153-1162). More recently, 2-TDC has been identified as a volatile organic compound (VOC) produced by rhizosphere bacteria belonging to the genera Burkholderia _r Serratia and Escherichia (Blom et al. 2011. Production of plant growth modulating volatiles is widespread among rhizosphere bacteria and strongly depends on culture conditions. Environ. Microbiol. 13: 3047-3058).

The ability of wild tomato species to produce significant amounts of 2-TDC has been linked to the high innate resistance of these plants against the attack of herbivorous insects and mites (Williams et al. 1980. 2- Tridecanone: A Naturally Occurring Insecticide from the Wild Tomato Lycopersicon hirsutum í.glabratum. Science 207: 888-889; Weston et al. 1989. Trichome secretion composition, trichome densities, and spider mite resistance of 10 accessions of Lycopersicon hirsutum. Journal of American Horticultural Science, 114, 492-498; Chatzivasileiadis and Sabelis (1997) Toxicity of methylketones from tomato trichomes to Tetranychus urticae Koch. Experimental and Applied Acarology, 21, 473-484; Gonc.alves, et al. 1998. Variation of 2-tridecanone level in tomato plant leaflets and resistance to two mite species {Tetranychus sp.). Euphytica, 104.33-38).

There are studies that demonstrate insecticidal activity and 2-TDC strokes (Antonious et al. 2003. Insecticidal and acaricidal performance of methyl ketones in wild tomato leaves. Bull. Environ. Contam Toxicol. 71: 400-407). It is known that the application of this methyl ketone does not induce phytotoxicity when sprayed at concentrations of 0.44 g per liter of water (Antonious. 2004. Persistence of 2-tridecanone on the leaves of seven vegetables. Bull. Environ. Contam Toxicol. 73 : 1086-1093). 2-TDC has been described as a mosquito and tick repellent (Roe (2004) Method of repelling insects. US Patent Application 20040242703, Kina code AI Myers, Bevel, Sibley, & Sajovec, Raleigh, NC.). The use of 1 or more methyl ketones, including 2-TDC, for the control of nematode infestations in plants as well as the construction and use of transgenic plants and genetically modified bacteria to be applied in plants capable of producing 2 has also been disclosed. -TDC or a combination of methyl ketones and related compounds for the control of attacks by insects and nematodes (Bradley et al. 2009, patent application WO 2009/100433 "Methods and compositions for plant pest control"; Bradley et al. 2011, application WO 2011/100650 "Improved compositions and methods for pathogen control in plants").

However, although 2-TDC has been shown to be effective in controlling pests (insects and nematodes) that attack plants, its effect on the control of bacterial infections has never been proven. Although the antimicrobial effect against certain bacteria and fungi of the essential oils of Glossocalyx staudtii has been described, some of which contain a high percentage of 2-TDC, as is the case with the extraction of the root of this plant where the 2- TDC constitutes 55.2% (Agnaniet et al. 2009. Chemical composition and antimicrobial activity of the essential oils of leaves, roots and bark of Glossocalyx staudtii. Nat. Prod. Commun. 4: 1127-1132), it has never been shown that Bactericidal effect of these essential oils is due to the presence of methyl ketone; In fact, the authors themselves suggest that this component of the oil is not as efficient as one might think (page 1130). In addition, there is at least one other bibliographic reference that could suggest that 2-TDC was not effective against bacterial infections, since it has been shown that it does not show antimicrobial activity against fungi or against Gram positive bacteria such as Staphylococcus _r Streptococcus _r or Bacillus ni Gram negatives such as Pseudomonas _r Escherichia _r or Proteus (Wood et al. 1995. Volatile ketones from interdigital glands of black-tailed deer, Odocoileus hemionus columbianus. J. Chem Ecol. 21: 1401-1408).

In short, bacterial infections can cause more or less serious diseases in plants and animals, including humans. The importance of prevention, control and, where appropriate, treatment of infections caused by pathogenic bacteria in humans is obvious. Equally important is to prevent and control the diseases caused by bacteria in animals since some of these infections can generate from large losses in the livestock sector, to food poisoning or even the transmission of the disease to man. On the other hand, plant diseases caused by bacteria or bacteriosis are responsible for significant losses in agriculture. Although phytopathogenic bacteria generally cannot cause disease in animals, in recent years evidence has accumulated that fruits, herbs, vegetables, and especially green leafy vegetables, are authentic reservoirs of enteropathogenic bacteria such as enteric Salmonella or Escherichia coli The origin of the infection can be diverse, with irrigation water being the main route of contamination. Since this type of food is usually consumed raw or with very little processing, the presence in them of this type of pathogens constitutes a risk to human health.

The control of animal and plant diseases caused by bacteria has been achieved primarily by chemical treatment with antibiotics. For the prevention of plant bacteriosis, copper-based chemical agents such as Bordeaux broth are also used. The negative effect that these compounds potentially have on both the environment and the health of animals and humans, as well as the development of resistance in pathogenic bacteria that make treatment very difficult or in the best case makes it more expensive, makes it convenient to search for safer alternative solutions. Few alternatives have been presented to date. The most striking case is that of 2-tridecanone, composed of wild tomato varieties, which gives them an innate resistance to attack by herbivorous insects and mites. This observation was the beginning that would lead later to demonstrate the insecticidal and acaricidal activity of 2-TDC, and to patent its use as i) mosquito and tick repellent, and ii) control of plant infestations by insects and nematodes. However, its effect on the control of bacterial infections has never been proven. In this sense, the present invention provides a new agent for the prevention and control of bacterial infections that cause diseases in both plants and animals and humans. The compound is 2-tridecanone (2-TDC), a long-chain aliphatic methyl ketone (Fig. 1), produced naturally by plants, animals and bacteria. Thus, the present invention offers a very advantageous alternative that also has application in the hygiene of facilities susceptible to contamination by bacteria.

In addition, this use of 2-tridecanone is unexpected and surprising, firstly because there are documents such as the one mentioned in the previous paragraph, which suggests a negative result in this type of treatment with 2-tridecanone. Secondly, as will be discussed later, because the control of bacterial infections occurs without bactericidal effect, that is, without destroying or attacking bacterial growth, but with its infective activity. General Description of the Invention

The present invention relates, in a first object, to a method for preventing and / or controlling bacterial infections in a plant and / or crop, characterized in that it comprises administering a long aliphatic chain methyl ketone of general formula (I):

(I)

where R ¹ represents a C9-C13 aliphatic chain, that is to say formed by a number between 9 and 13 carbon atoms, or what is the same R ¹ represents a CH ₃ (CH ₂ ) nCH ₂ chain where n = 7- ll,

as is 2-tridecanone (2-TDC). Within the scope of the present invention, the action of "controlling" is understood as avoiding additional infections to those already existing, it being not found that the compound in question has a treatment effect whereby already established infections are eliminated. As inferred from the above definition, the term "long aliphatic chain" refers in the present invention to a chain formed by a number comprised between 9 and 13 carbon atoms.

A second object of protection is constituted by a method to prevent and / or control infections caused by pathogenic bacteria of animals and humans that could be found contaminating food production facilities and food treatment, containment facilities, transport and care of animals, and in Medical materials and facilities likely to be contaminated with pathogenic bacteria, by applying the aliphatic long chain methyl ketones described above. A third object of protection consists in the use of the aliphatic long chain methyl ketones of general formula (I) defined above, such as methyl ketone 2-tridecanone, in the prevention and / or control of bacterial infections both in the phytosanitary field, such as in the field of Hygiene and Health of facilities, food or in the manufacture of consumable products, among other uses. A fourth object of protection refers to the aliphatic long chain methyl ketone of general formula (I) defined above, such as methyl ketone 2-tridecanone, for use in medicine, and more particularly particularly preferably to its use in prevention and / or treatment of bacterial infections in a subject. When methyl ketone is described herein, and more specifically 2-tridecanone, for use in medicine, and preferred embodiments of said methyl ketone, it should be understood that the invention also relates to or covers the use of methyl ketone in medicine.

It should be understood herein that an object on which 2-tridecanone or another aliphatic long chain methyl ketone of general formula (I) is applied is "susceptible to being contaminated by bacteria" when said object, be it a plant, an animal , a human being, a product or an installation presents a risk of contagion, for any reason (sensitivity, exposure ...). As derived from this section, the invention is based on the application / administration of the natural organic aliphatic long chain methyl ketone compound of general formula (I), with 2-tridecanone (2-TDC) being the best example, to individuals (plants , animals, humans), products or facilities at risk of bacterial contamination that may lead to the development of a disease. With it, it is primarily intended to reduce the adverse effects generated in the environment and in animal and human health of the classic treatments with contaminating chemical agents and antibiotics that can favor the emergence of resistant microorganisms. Thus, the invention is about the use of 2-tridecanone or other aliphatic long chain methyl ketone of formula (I) in the prevention and treatment of bacterial infections, both in plants, as in animals, humans and even facilities and Foods likely to be affected by bacteria contamination. Each of these applications is treated separately here, depending on the application object.

The main advantage of using aliphatic long chain methyl ketones defined by formula (I) is that they are natural compounds produced by numerous organisms, including plants and animals, of low or no impact on the environment. So far no harmful effects of these compounds have been described, especially 2-TDC on living organisms. This compound is available in the market and is not an expensive product (Alfa Aesar 83.6 euros / 100 g) ·

In addition, the methyl ketones described herein, specifically 2-TDC, are biodegradable (there are bacteria capable of using 2-TDC as a carbon source). All this implies that their use does not generate problems from an environmental point of view and is free of risks to human and animal health. This result is of double interest in the field of technology for the following reasons: first, it is unexpected because there are documents in the state of the art that suggest the absence of a significant and interesting effect of long chain methyl ketones aliphatic in bacteria and the infections they cause. Thus, Wood et al (1995; Volatile ketones from interdigital glands of black-tailed deer, Odocoileus hemionus columbianus. J. Chem Ecol. 21: 1401-1408) prove that 2-tridecanone does not show antimicrobial activity neither against fungi nor against Gram positive bacteria such as Staphylococcus _r Streptococcus _r or Bacillus nor Gram negative such as Pseudomonas, Escherichia, or Proteus. Secondly, the result obtained with the present invention is surprising, insofar as it has been found that the mechanism by which 2-TDC limits bacterial infection is not due to a bactericidal effect (consisting of destruction / death of bacteria). The fact that the growth of the bacteria is not affected in the presence of 2-TDC at certain concentrations, a compound that, on the contrary, if it limits the infection, reduces the selective pressure and therefore, the appearance of possible resistance, is a result totally unexpected, especially in view of what is indicated by other authors.

The investigations that have led to the present invention have made it possible to verify that indeed the addition of a long-chain aliphatic methyl ketone of formula (I), the best example being 2-TDC, at different concentrations (preferably in the range of 1 μΜ and 10 mM, and more preferably within the range of 5-500 uM) did not affect the growth of different bacteria (both bacteria that interact with plants such as Sinorhizobium meliloti (mutual)) and Pseudomonas syringae (pathogenic) or pathogenic bacteria of animals such as Salmonella typhimurium) . However, in all the cases tested, it did affect a bacterial behavior that is closely related to the virulence of pathogenic bacteria: swarming motility, a type of bacterial translocation that occurs on surfaces and that requires flagellar activity. Specifically, the addition of commercial 2-TDC (SIGMA) in concentrations of the micromolar order induced surface motility in bacteria that interact with plants (such as S. meliloti that forms nitrogen-fixing symbiosis with alfalfa plants (Fig. 2A), and in the phytopathogenic bacterium P. syringae (Fig. 2B)), while canceling the surface movement of a pathogenic bacterium of animals such as S. typhimurium (Fig. 2C). Similar results to those obtained with 2-TDC in the Swarming motility of S. meliloti and P. syringae have been obtained with methyl ketones 2-dodecadone, 2-pentadecanone, and 2-undecanone, results that demonstrate the potential of these methyl ketones for the uses described herein (Fig. 7 ). In addition, in some cases, the addition of 2- TDC not only altered swarming motility but also negatively interfered with the formation of biofilm (Fig. 3), another multicellular surface-associated phenomenon, showing connection with swarming motility, and which is responsible for the persistence of some bacterial infections. These results suggest that 2-TDC and other very similar methyl ketones can be recognized as a signal molecule in bacteria, interfering with phenotypes such as motility and biofilm formation that have a relevant role in the interactions that these microorganisms establish with eukaryotic hosts.

Detailed description of the invention

Preferably, the long aliphatic methyl ketone chain of general formula (I) is a linear aliphatic chain. The term "linear chain" refers in the present invention to a chain formed by a number comprised between 9 and 13 carbon atoms bonded together by C-C covalent bonds, complemented its structure with hydrogen bonds.

More preferably, the linear aliphatic chain is saturated. The term "saturated chain" refers in the present invention to a carbon chain in which there is no double or triple bond.

Preferably, the aliphatic long chain methyl ketone of general formula (I) is selected from the group consisting of 2-tridecanone, 2-dodecadone, 2-pentadecanone and 2-undecanone. In the most preferred embodiment, the methyl ketone is 2-tridecanone.

The use of the invention as a phytosanitary product It is of special interest, although not limited to the invention, in the application of the aliphatic long chain methyl ketones defined herein, such as 2-TDC, in plants or crops that do not produce significant amounts of these compounds during times of risk, to protect them from bacterial infections that can potentially cause them harm or disease. Since there are few plants in which the significant production of these compounds has been described, the invention is potentially useful for all types of plants.

The methyl ketone described can be administered in diluted form in a liquid. Preferably, it is administered as a solution. More preferably still, the aliphatic long chain methyl ketone of general formula (I) is diluted in the solution in a concentration between 1 μΜ and 10 mM, including both limits, and in the most preferred case is between 5 μΜ and 500 μΜ , including both limits. Also preferably, the solution containing the compound in question is an ethanolic solution. Said ethanolic solution is preferably 0.1% ethanol (v / v, ethanol / water), such that 1 L of solution containing the active compound methyl ketone at a concentration of 50 μΜ would contain 999 ml of water, 1 ml of ethanol and: 2-TDC mg previously dissolved in ethanol, or 8.5 mg of 2-undecanone; or 9.2 mg for 2-dodecanone; and 11.32 mg for 2- pentadecanone.

In a preferred embodiment, the aliphatic long chain methyl ketone of formula (I), such as 2-tridecanone, is applied by spraying (spray) the aerial part of the plant with the solution, which is preferably 0.1% ethanolic ( v / v, ethanol / water), containing effective concentrations of the compound. It is sprayed trying to make the surface to be treated homogeneously moistened.

In another preferred embodiment, the solution is injected directly into the plant stem, by methods conventional.

In another preferred embodiment, the invention involves the direct administration of the methyl ketone solution defined above to the soil or other plant growth medium. Administration can be indirectly through irrigation water or a nutrient solution for the plant, or by immersion of the root system or of the entire plant (or seeds) in the solution with 2- tridecanone or other methyl ketone long aliphatic chain of general formula (I). The solution would be prepared as indicated above, being able to replace the water with nutrient solution for plant growth. In any of the cases mentioned, the applications of the compound on the plant or the cultivation soil or other growth medium will be carried out in non-phytotoxic concentrations, preferably at times of the year of maximum risk, and several applications can be made spaced over time . The phytotoxic concentration to be used will depend on the type of plant and the mode of administration, since for example it has been found that at similar concentrations, in a hydroponic way the phytotoxic effect is more pronounced than in aerial spraying, always within the intervals defined in this memory.

Thus, the maximum effectiveness of the methyl ketone of general formula (I) was obtained using concentrations between 1 μΜ and 10 mM, including both limits, although obviously the optimum concentration of the compound to exert its protective effect depended on the type of plant, phase of its development, as well as the frequency and form of application. Thus, the effect of methyl ketone of general formula (I) on the limitation of bacterial infections at lower concentrations (minimum 4.4 times lower) than those described as non-phytotoxic after spray application has been demonstrated, which means a big advantage. Also, 2-TDC or other methyl ketone of general formula (I) can be applied alone or mixed with other additives known and commonly used in the field of Agriculture, for example organic and / or inorganic fertilizers, insecticides, nematicides, fungicides , bactericides and herbicides, and any combination thereof. Methyl ketone can be applied to plants before, after or simultaneously to the mentioned pesticides, herbicides or the like.

With regard to the method of prevention and / or control of bacterial infections by means of 2-tridecanone or another methyl ketone of general formula (I) for hygienic and sanitary purposes within the field of hygiene and food, it should be noted that the forms of administration and concentrations described for phytosanitary use are applied in the same way here. Thus, also in this case the effectiveness of methyl ketone, specifically 2-TDC, was observed using concentrations between 1 μΜ and 10 mM including both limits in a solution, preferably ethanol, and more preferably in a concentration between 5 μΜ and 500 μΜ, including both limits. Said ethanolic solution is preferably 0.1% ethanol (v / v, ethanol / water).

The product to be treated can be of a very diverse nature: food, surgical equipment ... or it can be an installation susceptible to contamination by bacteria (food production and treatment, containment, transport and care of animals, hospitals and health centers , kitchens, cooling systems or towers, humidifiers ...). This method is aimed primarily, but not limited to, products from the fruit and vegetable sector, in order to interfere with the mechanisms of infection of potential pathogens that were contaminating the edible plant material. However, this method can also be used in other products that may be contaminated, such as those of animal origin: eggshell ...

When it is a food product, such as fruit and vegetable products, the administration of the compound can be done after its collection, and always in concentrations not toxic to human consumption. In another preferred embodiment, the method is carried out by immersing the entire product in a solution of the active compound as described herein, in any of its variants.

As in the previous method, the recommended and most preferable form includes spraying the product intended for consumption or the relevant installation with an ethanolic solution containing concentrations between 1 μΜ and 10 mM or more specific, as described herein. . In another preferred embodiment, the air conditioning system and / or the cooling system of the installation to be treated is used to administer the solution containing the 2-tridecanone or other methyl ketone of the general formula (I). As stated in the previous section, the present invention is also directed to the use of methyl ketone of general formula (I), such as 2-tridecanone, for preventing and / or treating bacterial infections for phytosanitary purposes, that is in plants and / or crops. Preferably, the use of 2-tridecanone or another aliphatic long-chain methyl ketone is directed to the prevention and / or control of infections against Pvaudomonas syringae, P. savastanoi, Clavibacter michiganensis _r Xanthomonas campestris, X. citri, Xylella fastidiosa , Ralstonia solanacearum, Agrobacterium turnefaciens or Erwinia amylovora. Also contemplated here is the use of 2-tridecanone or other methyl ketone of general formula (I) to prevent and / or control infections caused by pathogenic bacteria of animals and humans that could be found contaminating food products and / or facilities for producing said foods. . More specifically, these products may be of vegetable origin, such as fruit and vegetable products, or of animal origin, such as eggs. The use of methyl ketone of general formula (I), such as 2-tridecanone, also covers the prevention of infections caused by bacteria that could be found contaminating animal containment and transport facilities, as well as medical facilities and hospital and the material used in them (for example, surgical material, catheters ...) or in other areas susceptible to contamination by bacteria, such as kitchens.

Another intended use for methyl ketone of general formula (I) is for the prevention of surface biofilms, by administration on said surface of a material.

Another object of the present invention is the use of 2-tridecanone or another methyl ketone of formula (I) as an additive against bacterial infections in oral hygiene products (dentifrices, mouthwashes) or body hygiene (hand soap).

With respect to the methyl ketone of formula (I) for use in medicine (or what is the same, the use of the aliphatic long chain methyl ketone of formula (I) for this purpose), it should be noted that the subject susceptible to being contaminated by Bacteria can be a human being or an animal. The 2-tridecanone or other aliphatic long chain methyl ketone is preferably administered in the form of a solution, this being more preferably an ethanolic solution. Preferably, the methyl ketone of formula (I) is contained in the solution in a concentration between 1 μΜ and 10 mM, including both limits, being more preferably between 5 μΜ and 500 μΜ, including both limits. Said ethanolic solution is preferably 0.1% ethanol (v / v, ethanol / water).

Preferably, the methyl ketone of general formula (I), especially 2-tridecanone, is administered against Salmonella typhimurium, enteropathogenic Escherichia coli, Proteus mirabilis _r Pseudomonas aeruginosa, Legionella pneumophila.

From this use of 2-tridecanone or another methyl ketone of general formula (I) in medicine it should be understood that the present invention also covers:

The use of the methyl ketone of formula (I) in the preparation of a pharmaceutical composition or a medicine for medicine, and more preferably for the prevention and / or control of bacterial infections; Y

- a method of prevention and / or control of bacterial infections in a subject by administration of said aliphatic long chain methyl ketone of formula (I). Such administration can be performed for example by topical application of open wounds, orally (to avoid gastrointestinal infections) or by inhalation devices (avoid respiratory infections), although it can be performed by any known and customary method in the field for this type of compounds. Administration is preferably carried out in the amounts and formats mentioned above: in a solution that is preferably ethanolic, at a concentration between 1 μΜ and 10 mM, including both limits, more preferably between 5 μΜ and 500 uM, including both limits, etc. .

Description of the figures

Figure 1.- Chemical structure of methyl ketone, 2- tridecanone (2-TDC).

Figure 2.- Effect of 2-TDC on the surface motility of bacteria that cause infections in plants and animals: Sinorhizobium meliloti, strains GR4 and GR4fadD; Pseudomonas syringae pv. syringae (Pss) strain BTlll and P. syringae pv. tomato (Pst) strain DC3000, and Salmonella typhimurium strains LT2 and 4155. In all cases the behavior of each bacterium without treatment (M) is shown, in the presence of 20 i of ethanol (M + E), or in the presence of 20 i of an ethanolic solution of 2-TDC (M + T). The concentration used in each case is indicated.

Figure 3.- Effect of 2-TDC on the biofilm formation capacity of P. syringae pv. syringae BTlll. The biofilm formed on the surface of a glass tube after growth in medium containing only ethanol (M + E) or medium containing 2-TDC (M + T) at a final concentration of 50μΜ, was detected by staining with violet crystal.

Figure 4.- Protective effect of 2-TDC on the development of the black spot of tomato. Development of leaf necrosis of tomato plants treated with the control mixtures C + Mb and E + Eb, and treated with 2 different concentrations of 2-TDC (T + MTlb and T + MT10b).

Figure 5.- Effect of the treatment of tomato plants with 2- TDC on the survival over time of the P. syringae pv tomato DC3000 bacteria in leaf tissue (expressed as colony forming units (cfu) per cm ² ). The symbols represent survival in plants treated with C + Mb (circle), E + Mb (rhombus), T + MTlb (square), or T + MT10b (triangle). Figure 6.- Inhibitory effect of 2-TDC on nodule formation in alfalfa plants by Sinorhizobium meliloti bacteria, strain GR4. Figure 7.- Effect caused by different methyl ketones on the surface motility of Sinorhizobium meliloti GR4 and Pseudomonas syringae pv syringae BT111. The graph shows the average surface dispersion halo of Sm GR4 (gray bars) and Pss BT111 (black bars) after 24 hours of incubation in the absence of methyl ketones (control), in the presence of ethanol (E), or in the presence of 2-undecanone (2- UDC), 2-dodecanone (2-DDC), 2-tridecanone (2-TDC) or 2- pentadecanone (2-PDC). The error bars represent the standard error and different letters indicate statistically significant differences according to an ANOVA test (P <0.05).

Examples

Example 1. Protective efficacy of the application of 2-TDC in the appearance of the black spot of tomato caused by the phytopathogenic bacterium Pseudomonas syringae pv. tomato DC3000. Tomato {Solanum lycopersicum "Moneymaker") was used as plant material. The seeds germinated and grew in seedbeds with composted peat in a growth chamber under controlled conditions of light, T ^a and humidity for 3 weeks. Subsequently, they were individually transplanted in plastic pots 14 cm in diameter and 13 cm high, stuffed with peat, where they remained 2 more weeks, before proceeding to infection with the bacteria.

As a phytopathogenic bacterium, the strain DC3000 of Pseudomonas syringae pv. tomato (Pst). The bacterium was grown at 28 ° C in solid LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 1.5% bacteriological agar), added rifampin (Rif) (10 mg / L). From these plates, a bacterial suspension was prepared in a solution of 10 mM MgCl ₂ resuspending bacterial mass until reaching an optical density (DC> 6oonm) of 0.3-0.4 (corresponds to approximately 10 ⁸ colony forming units (CFU) / me) . The chemical treatment with 2-TDC was carried out in 2 stages: i) Addition of 40 mg of 2-TDC (equivalent to about 26 kg of the compound / Hectare) to the substrate (peat) of each pot, 24 hours before the application of the pathogen and ii) foliar application by spraying two different concentrations of the compound (50iM and 5001M) at the same time of application of the pathogenic bacteria. It has been proven that these concentrations of 2-TDC do not generate appreciable adverse effects on bacterial growth in pure culture. Nor have harmful effects been observed in the development of the plant.

The following solutions were prepared for application to the substrate:

-T-solution: solution containing 2-TDC prepared as described: 40 mg of the compound was dissolved in 1 ml of ethanol (EtOH). Once the solution was achieved, the mixture was added to 24 ml of ¾0.

- Solution E, control solution containing EtOH in the same concentration as in Sol. T (4% EtOH (v / v)), and prepared by adding 1 ml of EtOH in 24 ml of ¾0. -Solution C, irrigation control consisting of 25 ml of ¾0.

The pots were watered with 25 ml of solution T, E or C that was applied around the plant stem. Immediately afterwards, an additional 25 ml of sterile water was applied to allow a good penetration of the treatments in the substrate.

In all cases, irrigation was carried out 24 hours before the application of the phytopathogen. For foliar application, the following mixtures were prepared:

-Mix M: 40 ml of a 10 mM MgCl ₂ solution -Me mix: 40 ml of M mixture containing 40 μΐ of ethanol. - MT1 Mix: 40 ml of mixture M containing 40 μΐ of a stock solution of 50 mM 2-TDC (prepared by dissolving 10 mg of 2-TDC per ml of EtOH).

-MT10 Mix: 40 ml of M mixture containing 40 μΐ of a stock solution of 500 mM 2-TDC (prepared by dissolving 100 mg of 2-TDC per ml of EtOH).

-Mb mix: 40 ml of a 10 mM MgCl ₂ solution containing the Pst DC3000 bacterium (10 ⁸ CFU / ml) prepared as indicated above.

-Meb mix: 40 ml of Mb mixture containing 40 μΐ of ethanol. - MTlb Mix: 40 ml of Mb mixture containing 40 μΐ of a 50 mM 2-TDC stock solution (prepared by dissolving 10 mg of 2-TDC per ml of EtOH).

- MTlOb Mix: 40 ml of Mb mixture containing 40 μΐ of a stock solution of 500 mM 2-TDC (prepared by dissolving 100 mg of 2-TDC per ml of EtOH).

In this example, 20 pots were used that underwent the following treatments:

24 hours before spraying the aerial part, the substrate was irrigated with Sol. T (10 pots), Sol. E (5 pots) and Sol. C (5 pots).

Before spraying the aerial part with the different mixtures, all the leaves of all the pots were sprayed with water to favor the opening of stomata. The pulverization of each of the mixtures was carried out by the beam and then 5 leaflets of 2 leaves of each plant, previously selected and duly marked.

Of the 10 pots irrigated with Sol. T, it was sprayed with: MT1 control mix (1 plant), MT10 control mix (1), MTlb mix (4) and MTlOb mix (4)

Of the 5 pots irrigated with Sol. E, it was sprayed with: ME control mixture (1 plant), and MEb mixture (4). Of the 5 pots irrigated with Sol. C, it was pulverized with: M control mixture (1 plant), and Mb mixture (4). The effect of each of the treatments on bacterial infection in tomato plants was determined by 2 approaches: evaluation of symptoms 10 days after the infection was performed, as well as by determination of bacterial survival over time by means of a CFU count . A first count was made 3 hours after infection to know the original inoculum and subsequently counts 3, 6, and 10 days after inoculation. For the CFU count of leaves infected with Pst DC3000, with the help of a punch of 1 cm in diameter, 5 discs were isolated per treated leaf, 1 for each of the pulverized leaflets (equivalent to a total leaf surface 3.9 cm ² ). The 5 discs were macerated and homogenized in 1 ml of sterile 10 mM Cl ₂ Mg, suspension of which serial dilutions were made that were seeded in LB RiflO plates. The process is repeated on the same sheet as many times as the size of it allows (3-6 times), and proceeds in the same way with the second sheet of the same plant that was also sprayed. Thus, for each treatment and time of infection the count of 6-12 replicas of which a mean and standard error is obtained is performed.

Fig. 4 shows the appearance of the plants after 10 days after performing the different treatments. Plants that were treated with 2-TDC (MTlb and MTlOb) developed much less necrotic spots on the leaves than control plants (Mb and Eb). The effect was also dose-dependent, since the plants treated with the more concentrated 2-TDC solution (MTlOb) showed a better appearance than those treated with a lower concentration dose, the development of necrotic spots being almost negligible. The 2-TDC treatment also had an effect on bacterial survival in plant tissues (Fig. 5). When the bacterium was applied in the absence of 2-TDC (with water or with ethanol), the population reached a maximum around 10 ⁷ colony forming units (CFU) per cm ² of leaf between 3 (water) and 6 ( ethanol) days after application, to subsequently decrease approximately an order of magnitude at 10 days. On the other hand, in plants treated with 2-TDC, the bacterial population never reached the density of 10 ⁷ CFU per cm ² , the treatment with 500 μΜ of 2-TDC being especially striking, which did not allow bacterial growth. The population density at the end of the experiment in plants subjected to this treatment was significantly lower compared to the others.

Therefore, treatment with 2-TDC protects tomato plants from the development of a disease caused by bacteria.

Example 2.- Application of 2-TDC in nutrient solution to reduce infection caused by Sinorhizobium meliloti bacteria in alfalfa plants. Alfalfa (Medicago sativa var. Aragon) was used as plant material. The seeds were surface sterilized and germinated as described in Olivares et al. (1980). The germinated seeds were placed in glass tubes (2 x 20 cm) containing a filter paper and 10 ml of nutrient solution for nitrogen-free plants (Rigaud and Puppo, 1975). After spending 2 days in the dark, the plants were moved to a growth chamber under controlled conditions of light, T ^a and humidity, in which they were incubated 1 week before proceeding to inoculation with the bacteria.

As a bacterium capable of infecting alfalfa, strain GR4 of Sinorhizobium meliloti was used. The inoculum was prepared by growing the bacteria in liquid TY medium (Beringer, 1974) to 30 ° C until reaching an optical density (DC> 6oonm) of 0.5-0.6 (approximately 5 x 10 ⁸ cfu / ml). This inoculum was diluted in sterile water to obtain an inoculum density of 5 x 10 ⁶ cfu / ml, suspension with which the inoculation of the plants would be performed.

After 9-10 days of placing the alfalfa seedlings in the tubes, the bacteria were inoculated, adding to each tube / plant 1 ml of the prepared bacterial suspension as previously mentioned.

In this example, the application of 2-TDC treatments or control treatments (ethanol only) was performed at the time of inoculation with the bacteria. For this, two stock solutions of 2-TDC were prepared using ethanol as solvent: at 25 mM (5 mg of 2-TDC / ml of ethanol) and 5 mM (1 mg of 2-TDC / ml of ethanol). 10 μΐ of one of these solutions were applied to the 10 ml of nutrient solution (1: 1000 dilution) in which the plants grow, leaving therefore the compound in a concentration of 25 μΜ and 5 μΜ, respectively.

The success of the bacterial infection was determined by performing a daily count of the nodules (organ induced and colonized by S. meliloti) developed by plant. The results obtained in 2 independent experiments are shown in Fig. 6 of this report. The data suggest that the application of ethanol at a concentration of 0.1% to the nutrient solution in which the plants grow can cause a delay in the appearance of nodules, as well as a significant reduction in the average number of nodules per plant, which does not However, it recovers over time, until reaching similar values to those obtained in plants in which no treatment has been applied. However, and interestingly, the application of the ethanolic solution containing 2-TDC causes a delay and a reduction in the number of nodules, effects that are significantly superior to those obtained with the control treatment, and also dose dependent. The application of the more concentrated solution of 2-TDC caused a delay of 4-6 days in the appearance of the first nodules, as well as a reduction of almost 50% in the number of nodules developed.

Similar results have been obtained by applying 2-TDC at a final concentration of 25 μΜ 2 and 4 days before carrying out the inoculation with the bacteria, as well as applying the product 2 days after the bacterial inoculation. On the contrary, the protective effect is not observable when the application is carried out 4 days after having put the bacteria in contact with the plant.

Example 3.- Comparison of the effect caused by 2-undecanone, 2-dodecanone and 2-pentadecanone methyl ketones with that generated by 2-tridecanone in the swarming motility of the Sinorhizobium meliloti GR4 and Pseudomonas syringae patovar syringae BTlll bacteria. To check if methyl ketones similar to 2-tridecanone can have the same effect, and therefore similar applications, surface motility tests of S. meliloti GR4 and P. syringae pv bacteria were performed. syringae BTlll, in the absence and presence of these compounds.

The surface motility test was basically performed as described in Nogales et al. 2010 BMC Genomics 11: 157, with the sole caveat that the semi-solid minimum medium (MM) was prepared by adding 0.6% Difco noble agar (BD) instead of 0.6% purified agar (Pronadisa). The plates were allowed to solidify and dry for 15 minutes in a laminar flow cabinet with the lid open before applying the inoculum. The solutions containing each of the methyl ketones were prepared by dissolving 50 μπιοΙθΞ of each compound in 1 ml of ethanol, that is, 8.5 mg of 2-undecanone (2-UDC), 9.2 mg of 2-dodecanone (2- DDC), 10 mg of 2-tridecanone (2-TDC) or 11.32 mg of 2-pentadecanone (2-PDC).

For the preparation of the inoculum, the bacteria were grown in liquid TY medium (Beringer, 1974) at 30 ° C until an optical density (DC> 6oonm) of 0.9-1 was reached. Under aseptic conditions, 1 mL of these cultures was taken and the cells were collected by centrifugation (2 min, 10,000 rpm in a microcentrifuge), discarding the cell-free culture medium. The cell precipitate was washed twice with 1 ml of liquid MM (Robertsen et al. 1981) and finally resuspended in 100 μΐ of the same medium. A 2 μΐ aliquot of this cell suspension was placed on the surface of the semi-solid MM (prepared as indicated above) and allowed to dry for 10 min in laminar flow cabinet with the lid open. After that time, 20 μΐ of ethanol or 20 μΐ of a solution containing the aliphatic long chain methyl ketone, prepared as indicated above, was placed on the plate lid. Nothing was applied in the control. The petri dish was closed, sealed with parafilm, and incubated at 28 ° C in the dark. After 24 hours of incubation, the dispersion diameter of the colony was measured. At least 3 technical replicas have been made.

The results obtained are shown in Fig. 7. It can be observed that except in the case of 2-PDC that did not promote motility in the Pss BT111 bacteria, the other methyl ketones induced surface motility in both Sm GR4 and Pss BT111, with 2-DDC and 2-TDC being the ones that caused largest colony dispersion halo. Since 2-UDC, 2-DDC and depending on the bacterium also 2-PDC seem to have a bacterial motility on the surface similar to that observed for 2-TDC, it is expected that the preventive effect of bacterial infections can be extrapolated to these other methyl ketones.

Example 4.- Application of 2-TDC on the surface of food for human or animal consumption to avoid diseases caused by pathogenic bacteria that are contaminating the food.

The food is collected, which in this case can be a fruit and vegetable product (fruits, vegetables, herbs ...) or an animal product such as eggs that can be contaminated with enteropathogenic bacteria such as Salmonella. After a water wash, the products are passed through a belt on which a rain of an aqueous solution containing 2-TDC is applied at a final concentration of 50-500 µM. Each liter of solution contains 999 ml of water, 1 ml of ethanol and 10-100 mg of 2-TDC previously dissolved in ethanol.

Alternatively to the tape method, food can be dipped in pails containing the same solution. Once impregnated, they are allowed to dry at room temperature before packing.

Example 5.- Use of inhalers containing 2-TDC in the prevention of bacterial respiratory diseases in patients at risk (immunosuppressed, recurrent infections ...)

Prepare a 2-TDC suspension with final concentration between 50-500 μΜ for inhalation through a pressure device.

Inhale the preparation 1 or 2 times a day.

Claims

RE IVI DICATIONS

1. A method to prevent and / or control bacterial infections in a plant and / or crop, characterized in that it comprises administering at least one aliphatic methyl ketone of general formula (I)

(I)

where R represents an aliphatic chain formed by a number between 9 and 13 carbon atoms.

2. The method according to the preceding claim, wherein the methyl ketone is selected from the group consisting of 2- tridecanone, 2-dodecadone, 2-pentadecanone and 2-undecanone.

3. The method according to any one of claims 1 or 2, wherein the methyl ketone is administered in a solution containing said methyl ketone in a concentration between 1 μ 1 and 10 mM, including both limits.

4. The method according to the preceding claim, wherein the methyl ketone is administered in a solution containing said methyl ketone in a concentration between 5 μΜ and 500 μΜ, including both limits.

5. The method according to any one of claims 3 or 4, wherein the solution is ethanolic.

6. The method according to any one of the preceding claims, wherein the methyl ketone is administered by an application method selected from the group consisting of: spraying the aerial part of the plant, direct injection into the plant stem, direct administration to the substrate of plant growth, administration through irrigation water or a solution nutritious, immersion of the root system or of the entire plant (or seeds) in a solution containing methyl ketone, and any combination of such procedures.

7. The method according to any one of the preceding claims, wherein the methyl ketone is administered mixed with additives selected from the group consisting of: organic and / or inorganic fertilizers, insecticides, nematocides, fungicides, bactericides and herbicides, and any combination thereof ; or it is administered before, after or simultaneously to the administration of said additives.

8. A method to prevent and / or control bacterial infections in food, food production and treatment facilities, containment, transport and care facilities for animals, medical materials and facilities, and hospitality materials and facilities, characterized in that it comprises applying at least an aliphatic methyl ketone of the general formula (I)

(I)

where R ¹ represents an aliphatic chain formed by a number between 9 and 13 carbon atoms.

9. The method of the preceding claim, wherein the methyl ketone is selected from the group consisting of 2- tridecanone, 2-dodecadone, 2-pentadecanone and 2-undecanone.

10. The method according to any one of claims 8 or 9, wherein the methyl ketone is administered in a solution containing said methyl ketone in a concentration between 1 μ 1 and 10 mM, including both limits.

11. The method according to the preceding claim, wherein the methyl ketone is administered in a solution containing said methyl ketone in a concentration between 5 µΜ and 500 µM, including both limits.

12. The method according to any one of claims 10 or 11, wherein the solution is ethanolic.

13. The method according to any one of claims 8 to 12, wherein the methyl ketone is administered by an application method selected from the group consisting of: spraying, immersion, or dispersion by air conditioning system and / or cooling system.

14. Use of at least one aliphatic methyl ketone of general formula (I):

(I)

where R ¹ represents an aliphatic chain formed by a number between 9 and 13 carbon atoms, in the prevention and / or treatment of bacterial infections in the phytosanitary field or the health and hygiene of food, materials and facilities susceptible to contamination by pathogenic bacteria.

15. Use of the preceding claim against pathovars of one of the species selected from the group consisting of Pseudomonas syringae, P. savastanoi, Clavibacter michiganensis _r Xanthomonas campestris, X. citri, Xylella fastidiosa, Ralstonia solanacearum, Agrobacterium turnefaciens _r Erwinia amylovo _r Erwinia amylovo _r Salmonella typhimurium, enteropathogenic Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa and Legionella pneumophila.

16. Use of at least one aliphatic methyl ketone of general formula (I):

(I)

where R ¹ represents an aliphatic chain formed by a number comprised between 9 and 13 carbon atoms, in the prevention of surface biofilms, by administration on said surfaces of a material.

17. Use of at least one aliphatic methyl ketone of general formula (I):

where R represents an aliphatic chain formed by a number between 9 and 13 carbon atoms, as an additive against bacterial infections in oral or body hygiene products.

18. Use of the preceding claim, wherein the product is selected from the group consisting of a dentifrice, a mouthwash and a hand fertilizer.

19. Use of at least one aliphatic methyl ketone of general formula (I):

(I)

where R ¹ represents an aliphatic chain formed by a number between 9 and 13 carbon atoms, in the preparation of a pharmaceutical composition or medicine for medicine.

20. Use according to the preceding claim, in the prevention and / or treatment of bacterial infections.

21. Use defined in any one of claims 14 to 20, wherein the methyl ketone is selected from the group consisting of 2-tridecanone, 2-dodecadone, 2-pentadecanone and 2-undecanone.

22. A methyl ketone alif general formula (I):

(I)

where R represents an aliphatic chain formed by a number between 9 and 13 carbon atoms, for use in medicine.

23. Methyl ketone according to the preceding claim, which is selected from the group consisting of 2-tridecanone, 2-dodecadone, 2-pentadecanone and 2-undecanone.

24. Methyl ketone according to any one of claims 22 or 23, for use in the prevention and / or control of bacterial infections in a subject.

25. Methyl ketone according to the preceding claim, wherein the subject is an animal or a human being.

26. Methyl ketone according to any one of claims 22 to 25, wherein the methyl ketone is applied in a solution containing said methyl ketone in a concentration between 1 μΜ and 10 mM, including both limits.

27. Methyl ketone according to the preceding claim, wherein the methyl ketone is applied in a solution containing said methyl ketone in a concentration between 5 μΜ and 500 μM, including both limits.

28. Methyl ketone according to any one of claims 26 or 27, wherein the solution is ethanolic.

29. Methyl ketone according to any one of the preceding claims, wherein methyl ketone is administered against one of the species selected from the group consisting of Salmonella typhimurium, enteropathogenic Escherichia coli, Proteus mirabilis _r Pseudomonas aeruginosa and Legionella pneumophila.