BE1030645B1 - METHOD FOR DETECTING MICROORGANISMS - Google Patents

Abstract

Use of a liquid sampling composition comprising one or more surfactants and one or more enzymes for the detection by immunology or genetics of bacteria chosen from Salmonella, Pseudomonas and/or Listeria.

Description

METHOD FOR DETECTING MICROORGANISMS

Technical area

The present invention relates to a method for detecting microorganisms, based on sampling of these microorganisms, then by immunological or genetic marking or involving the cultivation of these microorganisms, as well as on compositions allowing sampling compatible with the tests immunological or genetic.

Prior art

In many areas of activity such as the agri-food sectors, communities, medical and veterinary sectors, problematic contaminations due to the presence of microorganisms are observed very frequently.

Classically, in hospitals and in medical or veterinary offices, the presence of microorganisms is responsible for nosocomial diseases while in the food industry and in communities, these microorganisms are responsible for the degradation of perishable foods but also for the transfer of contaminants to consumers. products from, for example, a meat, fruit, vegetable or other production chain.

Nowadays, strict hygiene standards are imposed and it is therefore necessary to ensure that the number of microorganisms responsible for such contamination is kept below an acceptable threshold value, this acceptable threshold value being specific to each area. of activity.

Planktonic bacteria are a first example of contaminating microorganisms, these bacteria are free at the level of a liquid or solid substrate and pose a problem in themselves because they are able to directly contaminate any type of surface such as foodstuffs, medical tools , conveyor belts, storage tanks or even human patients or animals themselves.

However, today, it is widely recognized that the problems linked to contamination by microorganisms are all the more important as the latter form biofilms.

Indeed, in any type of industry and more particularly in the field of the food industry, biofilms form inevitably, given the richness of the surrounding environment.

Biofims are defined as a consortium of

Mmicroorganisms embedded in a matrix of extracellular polymeric substances. In other words, biofilms are viscous films which develop on all surfaces, following the adhesion of microorganisms to these surfaces and the secretion by them of polymers covering them, facilitating their adhesion to the surface and thus forming the extracellular matrix. Biofilms thus constitute a protective layer around microorganisms that is very resistant to chemical and thermal attacks, which makes their elimination very difficult using conventional biocides and also represents a recurring and important source of contamination of the surrounding environment.

This resistance is explained on the one hand by the matrix of extracellular polymeric substances which forms a physical barrier to the diffusion of biocidal molecules in the biofilm and, on the other hand, by the sessile state of the microorganisms and their capacity to very quickly exchange genes responsible for certain biocide resistance mechanisms with each other.

From then on, we easily understand that biofilms are very resistant and extremely varied structures, for example different strains of the same bacteria can form different biofilms, these biofilms being even more varied as they are composed of several different bacteria. In addition, bacteria exchange resistance genes with each other and the environment also influences biofilms, further accentuating their complexity and resistance.

In addition, the extracellular matrix of biofims can be identified when it is strongly developed but, in the majority of cases, biofim grows insidiously in facilities and its presence will only be detected during product quality analysis. finished.

Concerning the growth of the biofilm, this took place in a cyclical manner, comprising a growth phase during which the accumulation of microorganisms occurs and a detachment phase during which entire pieces of biofilms and microorganisms are detached by erosion and under the effect of their weight to contaminate surfaces, foodstuffs, medical tools, conveyor belts, storage tanks or even human patients, consumers or the animals themselves.

This should result in the industrialist stopping his production line and carrying out a cleaning cycle to eliminate the biofilm, representing many hours of work, resources deployed and loss of yield.

From all this, it appears that contaminating microorganisms and/or biofims constitute a real problem, particularly in the field of health care (hospitals, dental or medical practices), veterinary care and the agri-food industry.

This problem is all the more critical as microorganisms and/or biofims can involve bacteria responsible for potentially fatal infections in individuals, whether these bacteria are present in hospitals, veterinary practices or even in the food and beverage industry. are ultimately found on food products intended for consumption.

As indicated above, the problem of microorganisms and more particularly biofilms is twofold. On the one hand, conventional disinfectants and biocides are very often ineffective because they do not succeed in reaching the microorganisms protected by the extracellular matrix of the biofilm, this matrix being of complex and very variable structure and composition. On the other hand, a biofilm is generally mixed, that is to say it contains a multitude of different bacteria or the same bacteria but which are of different strains, which favors the propagation of resistance genes between bacteria. biofilm and thus makes their treatment very difficult or even ineffective.

Consequently, from one environment to another or from one sector of activity to another, it is quite common for the biofilms detected to be completely different. In order to detect biofilms, there are kits for detecting the presence of biofilms on surfaces or in more specific installations (water circuits, etc.), these kits (such as that disclosed in document EP2537601) make it possible to carry out selective staining of biofilms. It is therefore currently possible to determine areas where biofilms are present in order to eliminate them without knowing the precise nature of the microorganisms (bacteria) causing the targeted biofilm. as a result, compositions for eliminating biofilms comprising several enzymes are used without however knowing whether the enzymes used and possibly formulated in a detergent base

(see for example document EP2243821) are really adequate to act on a given biofilm. For this reason, current treatments are rather random and non-specific, which is economically unprofitable and can result in wasted time and considerable plant downtime.

Furthermore, techniques for sampling microorganisms from surfaces have been developed in order to characterize and/or quantify the populations of microorganisms present on surfaces and responsible for contamination. These sampling methods therefore make it possible to determine, on a surface, the different types (of strains) of bacteria and microorganisms present.

Among the methods for sampling microorganisms present on a surface, one of the references in this area is the Standard

ISO 18593:2004/F} which provides and defines horizontal methods for sampling techniques on surfaces (more specifically on surfaces encountered in the environment of the agri-food industries). The “cloth/sponge” method is an example of sampling techniques cited in this Standard, this method making it possible to search for and/or count microorganisms present on a surface. Briefly, according to this sampling method, it involves wetting the cloth/sponge with a quantity of diluent which is sterile physiological serum, sampling the surface in two perpendicular directions before introducing the cloth/sponge into a sterile container with the diluent. Subsequently, after possible storage of the sample, it is analyzed quantitatively and/or qualitatively.

Unfortunately, if such a method makes it possible to ensure a sample of free microorganisms on the surface of a substrate, it is-

i.e. a sample of planktonic microorganisms, it is found to be ineffective when the surface is contaminated by microorganisms having formed a biofilm. Indeed, as explained above, the microorganisms protected by the extracellular matrix of the biofilm, this matrix being of complex and very variable structure and composition and the biofilms being mixed, they are even more varied and complex as they are composed of several different bacteria or different strains.

In addition, it turns out that sampling techniques based on a support impregnated with water can have a beneficial effect on the development of microorganisms, even when taking a sample from a surface. Indeed, it is recognized that certain bacteria exhibit increased growth in the presence of water, the support soaked in water therefore promoting this growth during sampling but also after it since a thin film of water remains on the support treaty where the sampling took place. This can therefore promote the development of certain uncollected microorganisms, in particular when they are protected by a biofilm.

In order to overcome the disadvantages of water-impregnated sampling supports, we know from the state of the art the document DE10304331 which discloses an enzymatic preparation which can be used to eliminate biofilms from surfaces in the absence of biocides. This enzyme preparation comprises one or more enzymes from the group of polysaccharidases and/or proteases and optionally nucleases.

Unfortunately, even if the compositions according to document DE10304331 present a certain effectiveness in terms of collecting microorganisms, it appears that they are insufficient given the diversity and complexity of microorganisms and biofilms which can be present on a surface of an installation and that they do not allow a representative sampling of the bacterial populations which are nevertheless present there, that is to say that they only allow a weak coverage of the microorganisms.

Indeed, the biofilms and/or microorganisms present on a surface of an installation are on the one hand composed of a varied and complex population of microorganisms, that is to say different microorganisms but also these different

Microorganisms can still be of different strains, leading to different resistances but also the formation of varied, complex and different extracellular biofilm matrices, sometimes within the same surface of an installation.

There is therefore a real need to provide a composition which aims to overcome the disadvantages of the state of the art by allowing effective, representative and adequate sampling of populations of microorganisms, whether they are free but also under the form of a biofilm with a complex structure which can vary considerably from one sample to another. In addition, there is a need to provide a composition allowing the collection of microorganisms from different types of surfaces, in different environments, stable over time and which is compatible with the

Users or with the production chain, for example food in a food industry.

As such, patent application WO 2018/002013 describes a viscous aqueous solution comprising one or more enzymes and 8.4% surfactants for application on a horizontal or even vertical surface, with a view to prolonged impregnation of a fabric placed on this surface, so as to recover a Maximum of the micro-organisms possibly present while preserving their viability and quantifying their presence.

However, the sampling time is long and the components were selected with the aim of preserving the viability of the microorganisms, not to allow a downstream immunological test.

Finally, these sampling and sampling steps must be compatible with immunological detection tests, which implies strong constraints in terms of the reagents to be used.

Brief summary of the invention

A first aspect of the present invention relates to the use of a liquid sampling composition comprising one or more surfactants and one or more enzymes chosen from the group consisting of a protease, a polysaccharidase, a laccase, a lipase, a cellulase and a mannanase, for the detection of bacteria chosen from Salmonella (e.g. Salmonella enterica, Salmonella bongori), Pseudomonas (e.g. Pseudomonas aeruginosa) and/or Listeria (e.g.

Listeria monocytogenes) potentially present on a surface.

Advantageously, detection is done by immunology or by genetic testing and/or after culturing the bacteria.

Preferably, for this use, the sampling composition comprises surfactants present at a content of between 0.02% and 1% by weight relative to the weight of said sampling composition.

Preferably, in this Use, these surfactants (present in the sampling composition) comprise at least one anionic surfactant, preferably said anionic surfactant being chosen from the group consisting of alkyl ether sulfates, preferably a salt of laureth sulfate, alkyl sulfate , alkyloenzene sulfonate and soaps, preferably the surfactant(s) is (are) essentially one or more anionic surfactants.

Preferably, in this use, the sampling composition further comprises at least 15% of a stabilizing agent chosen from a polyol, preferably glycerol, and propylene glycol, relative to the weight of said sampling composition.

Preferably, in this use, the sampling composition comprises a protease being a serine protease.

An aspect related to the present invention relates to a kit for the detection of microorganisms chosen from Salmonella sp. and or

Listeria sp. potentially present on a surface comprising: - a sterile liquid sampling composition comprising one or more surfactants and one or more enzymes chosen from the group consisting of a protease, a polysaccharidase, a laccase, a lipase, a cellulase and a mannanose; - means for controlled application of said sampling composition and/or means for sterile storage of the sampling composition after its application.

Preferably, the sampling composition of this kit comprises surfactants at a content of between 0.02% and 1% by weight relative to the weight of said sampling composition.

Preferably, the surfactants of this sampling composition (which is part of the kit) comprise at least one anionic surfactant being chosen from the group consisting of alkyl ether sulfate,

preferably a salt of laureth sulfate, alkyl sulfate, alkylbenzene sulfonate and soaps.

Preferably the surfactants of this sampling composition (which is part of the kit) are (essentially) one or more anionic surfactants.

Advantageously, the sampling composition [which is part of the kit} further comprises at least 15% of a stabilizing agent chosen from a polyol, preferably glycerol, and propylene glycol, relative to the weight of said sampling composition.

Preferably, this kit further comprises a preservation solution comprising a polyoxyethylene sorbitan derivative, preferably polyoxyethylene sorbitan monooleate, and/or a phosphoglyceride derivative, preferably phosphatidylcholine, and/or a protein hydrolyzate.

Advantageously, in this kit, the sampling composition comprises a protease being a serine protease and, optionally, the preservation composition comprises a serine protease inhibitor, preferably PMSF.

Preferably, in this kit, the preservation solution comprises a protein hydrolyzate, preferably peptone, at a content of at least 10 g/L.

Preferably, in this kit, the preservation solution further comprises at least one osmotic agent chosen from the group consisting of sodium chloride, potassium chloride, potassium sulfate, tribasic sodium phosphate, dibasic sodium phosphate , monobasic sodium phosphate, preferably said at least one osmotic agent is sodium chloride.

Preferably, this kit further comprises means for specific detection and/or specific quantification by immunology or genetics of bacteria, preferably chosen from Salmonella (eg.

Saimonella enterica, Salmonella bongori}, Pseudomonas (e.g.

Pseudomonas aeruginosa) and/or Listeria (e.g. Listeria monocytogenes).

Detailed description of an embodiment of the invention

The inventors have noticed that many compounds used for effective sampling of microorganisms interfere with the various tests for detecting microorganisms, tests which involve a culture of microorganisms, and at least one of the tests for measuring the ATP generated, genetic testing and immunological testing.

These tests are important to qualitatively measure which microorganisms are present, but also to measure the variation in number over time on a given surface, which is an important parameter, in any case for industries such as food industries (in the sense large), or hospitals, or even canteens. Particularly with regard to dangerous microorganisms such as _ Listeria sp. or Salmonella sp.

To solve this problem, according to the invention the use of a sterile composition for sampling, removing and collecting microorganisms for the identification of microorganisms (e.g. Listeria sp. and/or Salmonella sp. ), comprising: - ONE or more surfactants representing at least 0.01% by weight relative to the weight of said composition and a maximum of 1.5%, preferably a maximum of 0.5%, or even 0.1% by weight of said composition,

this surfactant or surfactants comprising, or even consisting of (essentially) one (or more) anionic surfactant, - QU Minus a polyol and/or propylene glycol as stabilizing agent (and/or viscosing agent), representing at least 15 %, preferably at least 18%, by weight relative to the weight of said composition, - advantageously, at least one sequestering agent (di- or multi-valent cations), - at least two enzymes (in concentration capable of degrading the possible bacterial biofilms or stains), said at least two enzymes being chosen from the group consisting of a protease, a polysaccharidase, a laccase, a lipase, a cellulase and a mannanase, this composition preferably having (i) a pH comprised between 5 and 11 and/or (ii) a dynamic viscosity (20°C) of between 10 and 100 mPass; preferably, these at least two enzymes present in the composition are chosen and incorporated in a content so as not to interfere with the microbiological tests which will be carried out subsequently.

Advantageously, the sample composition has been sterilized by physical means, such as irradiation, for example with gamma rays or by electron beam or by sterilizing filtration.

Advantageously, the sampling composition further comprises a preservative content, such as (2-)phenoxyethanol, not exceeding 50 ppm. The preferred compositions advantageously have a lower concentration of preservatives, for example less than 20 ppm 2-phenoxyethanol, less than 10 ppm 2-phenoxyethanol, less than 5 ppm 2-phenoxyethanol (part per million: weight of the sum of preservatives (2-phenoxyethanol): total weight of the composition), a content below the detection limit and/or below 0.1 ppm.

This sampling composition is preferably for application to a surface, such as a surface used in

Industries, for example food industries, or hospitals or canteens.

The characteristics of the sampling composition have the advantage of providing a composition for sampling microorganisms, whether in planktonic form or in the form of complex and varied biofilms. This composition allows precise sampling of microorganisms.

The sample composition, comprising at least two enzymes chosen from the group comprising a (serine) protease, a polysaccharidase, a laccase, a lipase, a cellulase and a mannanase, advantageously makes it possible to effectively degrade and destructure the extracellular matrix of biofilms. and in particular a varied and complex population of biofims having different extracellular matrices thus making it possible to release the bacteria in order to sample them.

The contents of preservative agents (eg 2-phenoxyethanol) of the enzymes used in the sample composition are therefore advantageously known or measured. This advantageously makes it possible to choose enzymatic compositions relatively low in preservatives.

Incorporation of a (serine) protease into the sample composition is preferred. However, the inventors noticed (1) that proteases interfere with downstream microbiological tests, such as tests involving enzymes (PCR, luciferase) or antigen recognition (ELISA). Furthermore, the inventors noticed that many commercial batches of protease contained very high levels of preservative, and the inventors noticed that these agents interfered with several downstream microbiological tests, especially those involving the cultivation of microorganisms for their amplification. , in particular the inventors noticed that Listeria sp. was particularly sensitive to such agents.

In addition, the sequestering agent as well as the pH between 5 and 11 of the sampling composition advantageously makes it possible to form complexes with mineral ions which will be fixed in a form preventing their precipitation by the usual reactions and thus to stabilize in time the composition of the sample, particularly in view of the enzymes present in such proportions.

In the context of the present invention, the sequestering agent potentially present in the sampling composition is preferably a chemical substance having the capacity to form complexes with mineral ions which it fixes in a form preventing their precipitation by the usual reactions . By way of example, the sequestering agent may be ethylenediaminetetraacetic acid, glucono-delta-lactone, sodium gluconate, potassium gluconate, calcium gluconate, citric acid, phosphoric acid, tartaric acid, sodium acetate, sorbitol, a compound containing a phosphorus atom, or even carboxymethyl inulin (the sodium salt). Alternatively, the sequestering agent may be a phosphorus oxide such as a phosphonate, a phosphinate or a phosphate or their mixture, or a salt thereof, an amine or an amine oxide carrying at least, in its structure , a phosphine, phosphine oxide, phophinite, phosphonite, phosphite, phosphonate, phosphinate or phosphate functional group, alone or in combination, or a salt thereof.

In the context of the present invention, it has been determined that the sampling composition according to the invention comprises at least one anionic surfactant which represents at least 0.01% (preferably less than 1.5%, or even less than 1 %, or even less than 0.5% or even less than 0.1%) by weight relative to the weight of this composition, makes it possible in a particularly advantageous manner to remove and sample a representative and complete proportion of a population of microorganisms present on a surface, whether planktonic or in the form of biofims, while not problematically interfering with detection tests, including those specific for Listeria (culture, labeling and/or antibody detection , genetic identification, PCR, molecular biology).

Advantageously, among these anionic surfactants are foaming surfactants, this having the advantage on the one hand of removing and sampling the microorganisms and on the other hand of trapping the microorganisms sampled in the composition according to the invention in order to carry out a sample effective and representative of the population of contaminants on a surface.

Preferably the surfactants are chosen so that, overall, they have an HLB value of between 10 and 16.

Preferably, the at least one anionic surfactant in the sampling composition is a salt (e.g. sodium) of laureth sulfate (CAS number 9004-82-4). It is preferably sodium laureth-2 sulfate and/or sodium laureth-3 sulfate

Preferably, the sampling composition comprises at least 15%, preferably at least 18% by weight of a stabilizing agent, preferably glycerol.

Preferably, the sampling composition comprises at least 60% by weight of water, preferably at least 70% by weight, or even at least 75% by weight of water.

Thus, the composition comprising at least one anionic surfactant (low content) as well as at least one stabilizing agent is stable over time, while being compatible with microbiological tests, including those for the detection of Salmonella and

Listeria.

As a result, the sampling composition, unlike current compositions, makes it possible to obtain a truly representative and complete sampling of the contaminants present on a surface, for different types of surfaces and for different environments. This is particularly advantageous because, by having a representative and sufficiently complete sample of the microorganisms, they can subsequently be identified and a targeted and effective elimination treatment, using the appropriate compounds and no longer a cocktail of compounds very often. ineffective, or even ecotoxic, can be applied to the surface.

This of course represents a considerable economic and time advantage.

The interest in the sampling composition is therefore twofold in that it allows on the one hand to collect and sample all the contaminants from a surface whether they are planktonic or in the form of biofilms with a view to their analysis and then makes it possible to determine precisely what treatment to eliminate these contaminants will be applied.

Preferably, the sampling composition further comprises a pH regulating agent.

Preferably, the sampling composition has a pH of between 6 and 10, preferably between 7 and 9, preferably between 8 and 9, particularly advantageously between 8.2 and 8.6.

Preferably, the sampling composition is in liquid form, preferably in sprayable liquid form.

Preferably, the sample composition further comprises at least one additional enzyme chosen from the group consisting of oxidoreductases, lyases, transferases, esterases, glucosaminidases, nucleases. However, such additional enzymes are only preferred if they are present in compositions low in preservatives.

As explained above, the interest in the sampling composition is twofold in that it allows on the one hand to sample and sample all the contaminants from a surface whether they are planktonic or in the form of biofilms and that it then makes it possible to precisely determine which treatment to eliminate these contaminants will be applied after analyzes have been carried out to precisely identify and characterize the contaminants.

Consequently, it is important that the sampling composition (sampling), which contains the contaminants, microorganisms and biofilms following sampling, can allow a reliable, correct and representative analysis of the sampled population without said composition after sampling not interferes with subsequent analyses.

The invention therefore also relates to a kit for removing, sampling and preserving microorganisms in a living or revivable state for subsequent laboratory analyses.

This kit is useful for the detection of Listeria and/or Salmonella.

This kit includes the sampling composition described above, and a means of controlled application thereof. For example, a sprayer calibrated so as to project the same quantity of composition, with the same dispersion and in droplets of identical volume.

A volume to be sprayed is preferably 0.2 ml. A smaller volume (e.g. 0.1; 0.15 ml) or larger (0.25: 0.3; 0.4; 0.5 mi) can be chosen, as long as it is well calibrated. Preferably, the calibrated sprayer has a wide dispersion angle, which makes it possible to cover an area of 10 cm? with an application volume of 0.2 ml or less.

In other words, a kit is provided for removing (from a surface), sampling and preserving microorganisms in the living or revived state for subsequent laboratory analyzes comprising: - a sampling composition (detachment and/or sampling) sterile microorganisms as described above, - Qu less a device for controlled application of this sampling composition (detachment and sampling) on the surface; - preferably at least one means of sterile harvesting of these microorganisms, - preferably at least one sterile container, for example a sterile bag, advantageously comprising a solution for preserving the microorganisms in the living or revivable state for analysis of subsequent laboratory; - preferably means of detection by immunology or molecular biology (sequencing, PCR, specific nucleotide probes or primers) of bacteria, advantageously detection of Salmonella (e.g. Salmonella enterica and/or Salmonella bongori}, Pseudomonas (e.g. Pseudomonas aeruginosa ) and/or

Listeria (e.g. Listeria monocytogenes).

Preferably, the at least one means of sterile harvesting of the microorganisms of the kit according to the present invention is a tissue, a wipe or a sponge.

The preservation solution, in the context of the present invention, preferably comprises a protein hydrolyzate (peptone). and/or polyoxyethylene sorbitan and/or a phosphoglyceride derivative (e.g. lecithin); this composition preferably has the function of neutralizing any potential proteolytic and/or biocidal activity which would be present in the sample composition while ensuring the viability of the microorganisms recovered.

The invention further relates to a method for identifying and/or quantifying microorganisms comprising the following steps: at least one controlled application (spraying) of a described microorganism sampling, removal and sampling composition above, - allow this sampling composition (of removal, sampling) to act on this surface to be sampled, - deposit at least one means of harvesting these microorganisms on this surface to be sampled, - allow this means of harvesting to soak by this sampling composition (dropout, sampling),

- sterilely take this soaked means of harvesting microorganisms and place it in at least one sterile container, for example a sterile bag, comprising a solution for preserving microorganisms in a living or revivable state, - measure and/or quantify the presence of microorganisms by an immunological or genetic test (e.g. by sequencing and/or PCR), the microorganism(s) comprising Salmonella sp. and/or Listeria sp.

In the context of the present invention, an immunological test is preferably understood to be any test comprising one or more antibodies capable of binding specifically to a component of a specific bacteria. Such an immunological test is therefore qualitative (identity of the bacterial strain, presence of a given factor) and/or quantitative. This test may include secondary marking means, such as fluorescent, which allow rapid and precise analysis.

In the context of the present invention, a genetic test is preferably understood to be any test where the presence, or even the abundance, of a target nucleic acid molecule in the sample is analyzed.

The preferred genetic tests are sequencing and PCR (Polymerase

Chain Reaction} or other types of genetic amplification (including isothermal amplification techniques) involving probes and/or primers specific to the microorganism(s) to be identified (e.g.

Listeria and/or Salmonella).

Advantageously the bacteria to be detected are chosen from

Saimonella (e.g. Salmonella enterica, Salmonella bongori), Pseudomonas (e.g. Pseudomonas …—aeruginosa) and/or Listeria (e.g. Listeria monocytogenes).

Advantageously, the action step of the sampling composition takes place for a duration of at least 10 seconds, 20 seconds, 30 seconds, 1 minute, preferably at least 2 minutes, preferably at least 3 minutes, advantageously at least 4 minutes, particularly advantageously at least 5 minutes; preferably less than 15 minutes.

Preferably, the method according to the invention further comprises at least one step of storing this sterile container containing this soaked harvesting means, at a temperature lower than 15°C, preferably lower than 10°C, advantageously lower than 5 °C, which precedes the immunological test.

A related aspect of the present invention is therefore the use of the sampling composition or the kit comprising this sampling composition for the detection by immunology or genetics (eg by sequencing and/or PCR) of bacteria, advantageously chosen from

Salmonella (e.g. Salmonella enterica, Salmonella bongori), Pseudomonas (e.g. Pseudomonas aeruginosa)] and/or Listeria (e.g. Listeria monocytogenes).

Other characteristics and advantages of the present invention will be drawn from the non-limiting description which follows, and with reference to the examples.

Examples.

The first obstacles that the inventors had to overcome relate to the lack of representativeness of the biofilms generated in the laboratory, compared to the situation on site. This particularly complicates the choice of enzymatic activities (nature of enzymes, their abundances, presence of potentially deleterious compounds). On the basis of these preliminary tests, the inventors therefore decided that all the compositions which would be formulated in the laboratory should be tested in real conditions: many promising formulations on the laboratory scale are unusable, in practice, and the inventors have not found no published guidelines, even using their expertise, that could have helped them identify functional compositions.

However, once a complex formulation has been identified, such as those described below, certain compounds in this formulation can be replaced by others, which is easily testable in the laboratory (effectiveness, absence of toxicity for microorganisms). , then on site, or directly on site. Thus, to a certain extent, one protease is substitutable for another, in particular when these are serine proteases (subtilisins). However, when commercial enzymatic compositions are used, the inventors have noticed that, depending on the suppliers, and even the brands, different additives risk interfering with microbiology analyses, thus variations in the composition of the present invention are possible, thanks to the teaching of the present invention, but require following the rules of caution described in the present invention.

Example 1

A meat processing company located in Belgium was selected for this study. All facilities were previously cleaned and disinfected before taking samples.

The dropout protocol is applied with a limitation of mechanical actions that could introduce a reproducibility error; it includes the following steps:

Quickly scan (screen) the areas to be sampled with the UV lamp and BDK detection kit (Realco);

Preheat the sampling solution to 45°C;

Place two templates on the surfaces to be sampled (in order to delimit an area of 10cm by 10cm): physiologist water vs sampling composition (14 sprays to cover the area; 1.2 g/spray);

Leave the sampling solution to act for 5 minutes;

Place the sterile wipe on the two sampling areas and leave to soak;

Take sterilely using forceps and place in a sterile transport bag;

Store the samples in refrigerated containers at 4°C;

Afterwards evaluate the two zones with the lamp and the BDK.

A total of 6 sampling zones were included in this study: Zone 1 round table; Zone 2 lid for trash table; Zone 3 stainless steel tank; Zone 4 table saw; Zone 5 plastic surface; Zone 6 derinding.

In these 6 zones, the sample composition comprising enzymes (including a subtilisin, activity equivalent to 10

Anson units/kg} and detergents (approximately 10% by weight) allows a much better recovery of microorganisms, both in classical microbiology and by ATP measurement (2G) or by culture. Conversely, there were no more microorganisms remaining on the surfaces where the sampling solution was placed, unlike the surface treated with physiological water. The difference is especially marked at the level of classic microbiology, compared to ATP 2G or by test involving a culture step (little difference noticed).

The inventors reproduced the example, to include metagenomic analyses, while varying the contact time of the sample composition on the surface (30 seconds and 5 minutes). Again, the enzymatic composition allows for much better sampling (e.g. 100 times better in classic microbiology and 14 times better in qPCR). The bacteria found show an increased proportion in

Pseudomonas.

Example 2 - addition of a preservation solution;

Salmonella

The inventors, suspecting deleterious effects associated with the sample composition, sought to determine to what extent an adjuvant solution could counteract these, in the context of tests aimed at determining the presence of Salmonella and/or

Listeria, either by PCR or OPCR, or by using commercial detection kits, for example those from BioMérieux. To do this, a so-called preservation solution was tested (in g/l): Proteins (beef extract; 5); Peptone, 10; Polysorbate 80 (Tween 80; 5); Lecithin 0.7; NaCl, 5. In addition, various serine protease inhibitors have been used, with the aim of neutralizing the subtilisin activity of the sample solution.

The first series of tests was carried out in the laboratory, where known quantities of the bacteria (0.1 ml of 107 CFU/ml of Salmonella) were incubated either in a physiological solution (100 ml) or in the sample composition of example 1 (óml + 94 ml physiological water), or in the composition of example 1, before the preservation solution is added (6 ml sampling composition, 90 ml water, 4 ml of the preservation solution ).

When 107 CFU/ml of Salmonella are analyzed (therefore 104

CFU/ml in the final mixture), the different situations are compatible with the identification of the microorganism by means of PCR analyzes or the Vidas® Salmonella test. However, evidence by the test

Vidas SLM® (broth culture, then specific measurement by antigen test and fluorescence; this test provides information on the absence or presence of Salmonella) fails when the sample composition is present, whereas it works when the bacteria are diluted in physiological water or when the preservative solution has been added.

Furthermore, in this example, the inventors consider that the concentrations of Salmonella are high, which does not necessarily, or even rarely, represent the situation in the field, where even 1 CFU must be detected.

Example 3 - addition of a preservation solution; Listeria

In this case, the enzymatic solution causes problems in detection (enumeration and gPCR); these problems are reduced with the preservation solution, however the commercial kit

Vidas® (LMX) still does not work.

In addition, when the quantity of Listera inoculated is reduced, the microorganism is generally no longer detected following mixing (Vidas LMX®, never; Vidas LMO2®, not every time) with the sampling composition, while detection remains robust when the mixing was carried out only in physiological water. In this case (deliberately extreme), the conservation composition did not improve detection. As a precaution, the tests were reproduced and three strains of Listeria were tested with always the same results; these are strains ATCC 19114, NCTC 11994 and ATCC 13932.

By comparing all the results, the inventors conclude that the sample composition slows down the growth of microorganisms (Listeria), a problem which is partially resolved by the conservation composition. On the other hand, the sample composition interferes with certain downstream microbiological tests. Since the nature and abundance of microorganisms on a surface to be tested is unknown in advance, the inventors therefore conclude that the compositions of examples 1 to 3 are useful, but would benefit from being further improved.

Example 4 - Identifications of deleterious compounds

The inventors searched the various commercial enzymes to see if certain compounds interfered with downstream microbiological tests. The inventors have noticed that the presence of compounds such as phenoxyethanol, potassium sorbate or thiazolinone derivatives is quite widespread. Thus, the inventors have selected enzymatic compositions which are devoid of such compounds, or as poor as possible, even if the inventors have found significant concentrations of 2-phenoxyethanol in numerous commercial enzymatic compositions. Among the compounds sought are Bronopol, triclosan, idocarb, chlorhexidine, DCOIT, BIT, OIT, CIT, MIT, ethyl-paraben, methylparaben, butythylparaben, phenylparaben, isoproylparaben, isobutylparaben, propylparaben, salisyllic acid, 4-hydroxybenzoic acid, 4-methylbenzoic acid, benzoic acid, sorbic acid, dehydroacetic acid, 1-phenoxypropane-2 -ol, 1,2dioromo-2,4-dicyonobutane, 2-

hydroxybiphenyl, 2-phenoxyethanol (most abundantly found, sometimes up to 0.4% by weight), bensyl alcohol and chlorophenesin.

Comparative example 1

Suspecting an interference between the proteases of the sample composition and the enzymes or antibodies of the downstream tests, the inventors tested the effect of the addition of protease inhibitors in the preservation solution (see example 3), while avoiding as much as possible commercial enzymatic compositions comprising compounds identified as deleterious (see example 4).

Different ways of applying the protease inhibitor were tested. The chosen protease being a subtilisin or trypsin, two serine protease inhibitors were tested: PMSF and AEBSF.

PMSF provided good inhibition of the proteolytic activities of the serine proteases tested. On the other hand, AEBSF only inhibited trypsin. Measuring tests where Listeria (the 3 strains above were tested) is brought into contact with a composition comprising a subtilisin, then AEBSF in sufficient concentration to cause complete inhibition of the subtilisin have shown that different microbiological tests gave lower values, or even did not detect Listeria under the test conditions, unlike the control without enzymes or when PMSF is added.

Thus, the inventors chose to incorporate PMSF into the preservation solution to be tested, despite the disadvantages of this molecule.

Unfortunately, even under these conditions, the Vidas® test

LMX does not demonstrate the presence of Listeria monocytogenes.

Example 5 – Optimization of the sample composition

The inventors then decided to test the effectiveness of a highly diluted sampling composition and avoiding as much as possible the compounds identified as deleterious. In addition, enzymatic activities are reduced, as is the detergent content. Detergents are chosen after laboratory testing for their compatibility with microorganism life, including Listeria, which has been identified as more sensitive.

First of all, with detergent concentrations of 10% by weight, even in the presence of low enzymatic activity, in the presence of minimal preservative agent contents (e.g. non-detectable for almost all; phenoxyethanol less than 4 ppm ) and in the absence of

PMSF, Listeria detection tests, such as Vidas®, do not work well (massive underquantification), or not in a reproducible manner.

Even less specific tests do not work when a Listeria culture is subjected to these different treatments.

Thus, all the parameters of the composition must be checked, with the problem that too high a dilution of the enzymatic activities risks no longer allowing the microorganisms to drop out, while too high a dilution of the detergents risks no longer allowing application. homogeneous composition or good contact with the biofilm. Thus, the inventors noticed that, in addition to the quantity of the components, their nature had to be reconsidered.

A diluted formulation was tested comprising approximately 20% by weight of glycerol, approximately 0.03% by weight of an anionic detergent of the (sodium) laureth sulfate type, 0.4% by weight of sequestrant (carboxymethylinulin); 0.4% by weight of sodium glycolate; 0.04% by weight of a subtilisin (activity 1 Anson Unit}, as well as a lipase; an amylase, a cellulase and a mannanase. The inventors have selected enzymes, here commercial enzymes, devoid of preservative agents such as potassium sorbate, MIT, OIT, BIT and CIT.

When possible, the inventors used commercial enzymes devoid of phenoxyethanol (cellulase and mannanase), or with a low content of this molecule (subtilisin; 4 g/kg of the enzyme formulation, lipase; 0.249 g/kg and amylase; 0.233 g/kg). Enzymes other than subtilisin were added in weight contents of between 0.1 and 0.5% (weight of the enzyme composition: total weight) and the quantity of enzyme is typically 1 to 10% by weight relative to to commercial formulation.

Following the use of this composition, the presence of

Listeria is detected, and the Vidas® LMX test for Listeria is, finally, positive and reproducible. The inventors further noted that, under these diluted conditions, the addition of the protease inhibitor PMSF is not necessary, and even reduces the spread of Listeria.

The inventors consider that the concentration of enzymes other than proteases (here, a subtilisin) can still be reduced somewhat, e.g. the lowest concentration which allows detachment, just like those of the sequestering agent. In light of the present invention, this can be tested in the laboratory on the 3 strains of Listeria or in other models, including, preferably, more complex ones.

The inventors then tested a possible synergy with the preservation solution (see examples 2 and 3): given that the results are already very good, the improvement associated with the addition of the preservation solution is marginal, but it has was however interesting in the case of a test which includes propagation followed by detection by PCR.

It is of course understood that the present invention is in no way limited to the embodiments described above and that many modifications can be made without departing from the scope of the appended claims.

Claims (15)

1. Use of a liquid sampling composition comprising one or more surfactants and one or more enzymes chosen from the group consisting of a protease, a polysaccharidase, a laccase, a lipase, a cellulase and a mannanase, for the detection of bacteria chosen from Salmonella (e.g. Saimonella enterica, Salmonella bongon). Pseudomonas (e.g. Pseudomonas aeruginosa) and/or Listeria (e.g. Listeria monocytogenes) potentially present on a surface. 2. The use according to claim 1, in which the detection is done by immunology or by genetic testing and/or after culturing the bacteria. 3. The use according to claim 1 or 2, in which the sampling composition comprises surfactants present at a content of between 0.02% and 1% by weight relative to the weight of said sampling composition. 4. The use according to claim 3, in which the surfactants comprise an anionic surfactant, preferably said anionic surfactant being chosen from the group consisting of alkyl ether sulfate, preferably a salt of laureth sulfate, alkyl sulfate, alkylbenzene sulfonate and the soaps, preferably the surfactants are essentially one or more anionic surfactants. 5. The use according to any one of the preceding claims, in which the sampling composition further comprises at least 15% of a stabilizing agent chosen from a polyol, preferably glycerol, and propylene glycol, relative to the weight of said sample composition. 6. The use according to any one of the preceding claims, wherein the sampling composition comprises a protease being a serine protease. 7. Kit for the detection of microorganisms chosen from Salmonella sp. and/or Listeria sp. potentially present on a surface comprising: - A sterile liquid sampling composition comprising one or more surfactants and one or more enzymes chosen from the group consisting of a protease, a polysaccharidase, a laccase, a lipase, a cellulase and a mannanase; - means for controlled application of said sampling composition and/or means for sterile storage of the sampling composition after its application. 8. The kit for the detection of microorganisms according to claim 7, in which the sampling composition comprises surfactants at a content of between 0.02% and 1% by weight relative to the weight of said sampling composition. 9. The kit for the detection of microorganisms according to claim 8, in which the surfactants comprise at least one anionic surfactant, preferably said anionic surfactant being chosen from the group consisting of alkyl ether sulfates, preferably a salt of laureth sulfate, alkyl sulfate, alkylbenzene sulfonate and soaps, preferably the surfactants are essentially one or more anionic surfactants. 10. The kit for the detection of microorganisms according to any one of preceding claims 7 to 9, in which the sampling composition further comprises at least 15% of a stabilizing agent chosen from a polyol, preferably glycerol, and propylene glycol, relative to the weight of said sampling composition. 11.The kit for the detection of microorganisms according to any one of preceding claims 7 to 10 comprising a preservation solution comprising a polyoxyethylene sorbitan derivative, preferably polyoxyethylene sorbitan monooleate, and/or a phosphoglyceride derivative, preferably phosphatidylcholine , and/or a protein hydrolyzate. 12. The kit for the detection of microorganisms according to any one of preceding claims 7 to 11, wherein the sampling composition comprises a protease being a serine protease. 13.The kit for the detection of microorganisms according to claim 11 or 12, in which the preservation solution comprises a protein hydrolyzate, preferably peptone at a content of at least 10 g/L. 14. The kit for the detection of microorganisms according to any one of preceding claims 11 to 13, in which the preservation solution further comprises at least one osmotic agent chosen from the group consisting of sodium chloride, potassium chloride, potassium sulfate, tribasic sodium phosphate, dibasic sodium phosphate, monobasic sodium phosphate, preferably said at least one osmotic agent is sodium chloride. 15. The kit for the detection of microorganisms according to any one of preceding claims 7 to 14 further comprising means for specific detection and/or specific quantification by immunology or genetics of bacteria, preferably chosen from Salmonella (e.g. Salmonella enterica, Salmonella bongori)., Pseudomonas (e.g. Pseudomonas aeruginosa) and/or Listeria (e.g. Listeria monocytogenes).