Method for reducing the bacterial load on or in an air conditioning system

Abstract

The invention relates to a method for reducing the bacterial load on or in an air conditioning system, wherein the method has the steps of applying bacteriophages or a component thereof onto one or more parts of the air conditioning system. The invention further relates to the use of bacteriophages or a component thereof for reducing the formation of bacteria on or in an air conditioning system, for preventing the formation of a biofilm or for removing a biofilm on or in an air conditioning system, and / or for preventing the formation of odors in an air conditioning system. Finally, the invention also relates to specific bacteriophages.

Description

[0001] Methods for reducing the bacterial load on or in an air conditioning system

[0002] AREA OF INVENTION

[0003] The present invention relates to a method for reducing the bacterial load on or in an air conditioning system, as well as various uses of bacteriophages or a component thereof. Furthermore, the present invention relates to certain deposited bacteriophages as such.

[0004] BACKGROUND

[0005] During normal operation of an air conditioning system, such as a vehicle's air conditioning system, dirt particles and microorganisms often accumulate on the system's components after only a short time, especially directly on the evaporator and the pollen filter housing. These microorganisms use the dirt particles and airborne components as a food source and can form a stable biofilm. Heavy biofilm growth produces unpleasant odors that are clearly noticeable in the passenger compartment. This occurs particularly when air conditioning units are left unused for extended periods after the introduction of microbially digestible substances, especially in high humidity and mild temperatures.

[0006] Various products are available on the market to combat these odors. The range extends from simple spray products that temporarily mask odors to professional applications that use a combination of mechanical and chemical cleaning power to work directly at the source of the contamination and eliminate the odor-causing microorganisms. The following conventional systems are used:

[0007] - Pressure cup gun with rigid probe

[0008] An alcohol-based cleaner is sprayed directly onto the evaporator at high pressure using a pressure cup gun and a rigid probe. This removes dirt and deposits. The high spray pressure flushes the evaporator and removes the biofilm formed by microorganisms. This system is currently the most effective system for cleaning air conditioning systems.

[0009] - Self-emptying aerosol (click can)

[0010] An aerosol can placed in the footwell of a vehicle empties itself automatically after the spray nozzle is activated. The spray mist is then circulated through the air conditioning system via the recirculation setting.

[0011] - Aerosol foam with plastic probe

[0012] The foam is sprayed towards the evaporator using a probe, either through the air conditioning system's condensate drain or through the pollen filter box opening. The foam is intended to coat the evaporator and thereby clean / disinfect it.

[0013] - Ultrasonic nebulization

[0014] Here, a cleaning agent is atomized using an ultrasonic device placed inside the vehicle. The spray is then circulated through the air conditioning system via the recirculation setting and distributed throughout the interior.

[0015] - Suction cup gun with plastic probe

[0016] In the suction cup gun, air and cleaning agent mix through the Venturi effect. Similar to the pressure cup gun, the cleaning agent is applied directly to the evaporator. However, the flexible plastic probe prevents targeted flushing of the evaporator.

[0017] - Ozone generator

[0018] In this process, ozone is produced from atmospheric oxygen and then introduced into the vehicle interior. Through its decomposition mechanism, which has a high oxidative and reactive effect, organic compounds such as bacteria and other germs are broken down. The ozone emitted from the generator is hazardous to health and, if inhaled, can lead to lung damage and / or headaches, among other things.

[0019] Conventional systems typically use classic cleaning agents based on alcohols, surfactants, and / or biocides. However, the biocidal active ingredients, which are strictly regulated by the Biocidal Products Regulation (Biocidal Products Regulation), are potentially hazardous to the environment and to the health of humans and animals.

[0020] It can therefore be concluded that in state-of-the-art products for combating bacterially caused odors in air conditioning systems, little attention is paid to environmental and health aspects. Instead, commercial and functional considerations take precedence.

[0021] There may therefore be a need to overcome the problems and disadvantages of the current state of the art described above. In particular, there may be a need to replace chemical, environmentally harmful substances with biological raw materials that are harmless to humans and environmentally neutral.

[0022] It is known that bacteria can be combated by viruses, in particular by lysis and thus killing them. Such viruses, which are specialized to infect bacteria as host cells, are called bacteriophages and are classified into different groups according to their host specificity.

[0023] To date, bacteriophages are used only in the medical, therapeutic, and food technology sectors. These applications primarily involve medically relevant bacteriophages, meaning those that lyse pathogenic bacteria. For example, WO 2009 / 090097 A1 describes the use of bacteriophages in hygiene and care products, particularly for wound care. WO 2010 / 090542 A2 describes the therapeutic treatment of bacterial infections using specific bacteriophages. WO 2019 / 136108 A1 describes the use of bacteriophages for the treatment of Pseudomonas infections. DE 601 06 727 T2 discloses food packaging containing bacteriophages.

[0024] The use of bacteriophages in air conditioning systems, such as those found in vehicles, has not yet been described to date. However, given the adverse effects of microorganisms like bacteria on air conditioning systems, such as biofilm formation and unpleasant odors, bacteriophages represent a promising biological approach that can overcome the disadvantages of known chemical and environmentally harmful substances.

[0025] SUMMARY OF THE INVENTION

[0026] One object of the present invention is therefore to achieve a biological removal or at least reduction of bacteria in air conditioning systems without the disadvantages of the prior art due to chemical, environmentally harmful substances.

[0027] This problem is solved according to the invention by using bacteriophages or a component thereof to reduce the bacterial load on or in an air conditioning system, to prevent the formation of a biofilm or to remove a biofilm on or in an air conditioning system, and / or to prevent the formation of odors in an air conditioning system.

[0028] The inventors of the present invention have conducted extensive studies and have determined that vehicle air conditioning systems primarily contain representatives of the two bacterial genera Sphingomonas spp. and Methylobacterium spp. Furthermore, the inventors of the present invention were able to isolate suitable bacteriophages with these bacterial species as host organisms and thus selectively combat the bacteria predominantly present in a vehicle air conditioning system. A long-term effect (depot effect) and good material compatibility can also be achieved.

[0029] The present invention relates accordingly to a method for reducing the bacterial load (or a method for removing, killing or reducing the number of bacteria, preventing the formation of a biofilm or removing a biofilm and / or preventing the formation of odors) on or in an air conditioning system, wherein the method comprises applying bacteriophages or a component thereof to one or more parts of the air conditioning system.

[0030] Furthermore, the present invention relates to the use of bacteriophages or a component thereof for reducing the bacterial load (for removing, killing or reducing the number of bacteria) on or in an air conditioning system.

[0031] Furthermore, the present invention relates to the use of bacteriophages or a component thereof to prevent the formation of a biofilm or to remove a biofilm on or in an air conditioning system.

[0032] Furthermore, the present invention relates to the use of bacteriophages or a component thereof to prevent the formation of (bacterially caused, unpleasant) odors in an air conditioning system.

[0033] Furthermore, the present invention relates to selected bacteriophages or a component thereof, comprising at least one from the group consisting of Bacteriophage SP-P_L_l_2022, filed under DSM 34460, and Bacteriophage SP-P_M_l_2022, filed under DSM 34461. Further objectives and advantages of embodiments of the present invention will become apparent from the following detailed description.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] Further details of the present invention and other embodiments thereof are described below. However, the present invention is not limited to the following detailed description, which merely serves to illustrate the teachings of the invention.

[0036] It should be noted that features described in connection with one exemplary embodiment can be combined with any other exemplary embodiment. In particular, features described in connection with one exemplary embodiment of a method according to the invention can be combined with any other exemplary embodiment of a method according to the invention, as well as with any exemplary embodiment of a use according to the invention and with any exemplary embodiment of a bacteriophage according to the invention, and vice versa, unless expressly stated otherwise.

[0037] When a term is used with an indefinite or definite article, such as "ein," "eine," "eines," "der," "die," and "das" in the singular, this also includes the plural form, and vice versa, unless the context clearly indicates otherwise. The expressions "aufweisen" and "umfassen," as used here, do not only include the meaning of "contain" or "include," but can also mean "consist of" and "essentially consist of."

[0038] In a first aspect, the present invention relates to a method for reducing the bacterial load on or in an air conditioning system. The term "reduction of bacterial load," as used herein, which can also be referred to as "reduction of bacterial contamination," means in particular that the number of (live or active) bacteria is reduced, or that bacterial infestation is reduced or even prevented. For this purpose, the bacteriophages used, or a component thereof, may exhibit an antimicrobial effect or efficacy.

[0039] In the context of this application, an “antibacterial effect” or “antibacterial efficacy” is understood to mean the ability to kill bacteria or at least to inhibit their growth.

[0040] to control or limit reproduction. According to an exemplary embodiment, within the scope of the present application, an "antibacterial effect" or "antibacterial efficacy" is understood to mean a bacteriostatic and / or a bactericidal effect or property.

[0041] According to an exemplary embodiment, the method for reducing the bacterial load on or in an air conditioning system can thus be a method for removing, killing or reducing the number of (living) bacteria on or in an air conditioning system.

[0042] According to an exemplary embodiment, the method for reducing the bacterial load on or in an air conditioning system can be a method for preventing the formation of a biofilm on or in an air conditioning system, or a method for removing a biofilm from an air conditioning system. For the purposes of this application, a "biofilm" is understood to mean, in particular, a group of microorganisms, especially bacteria, in which the cells adhere to one another on a surface.

[0043] These adhering cells can be embedded in a self-produced matrix of extracellular polymeric substance, which can also be described as mucus. According to an exemplary embodiment, the method for reducing the bacterial load on or in an air conditioning system can be a method for preventing the formation of (bacterially caused, unpleasant) odors in an air conditioning system. For the purposes of this application, a "bacterially caused odor" is understood to mean, in particular, an odor produced by odorants generated by bacteria. For the purposes of this application, an "unpleasant odor" is understood to mean, in particular, an odor that is perceived as unpleasant by at least 50% of people, and especially by at least 80% of people.Sphingomas and Methylobacteria bacteriophages have proven particularly suitable for preventing the formation of unpleasant odors in an air conditioning system.

[0044] An air conditioning system can refer in particular to the air conditioning system of a vehicle, such as a motor vehicle, especially an automobile (e.g., with a gasoline or diesel engine, including a hybrid vehicle, or an electric vehicle), a commercial vehicle (e.g., a truck or a construction vehicle), a ship, or an aircraft. However, an air conditioning system can also refer to a stationary air conditioning system, such as the air conditioning system of a building.

[0045] The procedure involves applying bacteriophages or a component thereof to one or more parts of the air conditioning system.

[0046] The term "bacteriophage," as used here, refers specifically to a virus that can infect a bacterium, replicate at its expense, and ultimately lyse (dissolve) it. A bacteriophage usually infects one or more specific bacteria and therefore typically exhibits a corresponding host specificity. The delivered component of a bacteriophage may, in particular, be an antibacterial component of the bacteriophage.

[0047] According to an exemplary embodiment, the bacteriophages, or a component thereof, comprise at least one from the group consisting of Sphingomonas bacteriophages and Methylobacterium bacteriophages. In particular, the following bacterial species have been identified in vehicle air conditioning systems: Sphingomonas spp. and Methylobacterium spp. Therefore, Sphingomonas bacteriophages and Methylobacterium bacteriophages are especially suitable for reducing the bacterial load on or in an air conditioning system.

[0048] According to an exemplary embodiment, the bacteriophages, or a component thereof, comprise at least one from the group consisting of Bacteriophage SP-P_L_l_2022, deposited under DSM 34460, and Bacteriophage SP-P_M_l_2022, deposited under DSM 34461, or a component thereof. These bacteriophages have proven to be particularly suitable for reducing the bacterial load on or in an air conditioning system.

[0049] According to an exemplary embodiment, the application of bacteriophages or a component thereof comprises the application of a solution or a dispersion (in particular a suspension or an emulsion) containing the bacteriophages or a component thereof. Advantageously, the bacteriophages or a component thereof can be applied as a solution, for example, by dissolving the bacteriophages or a component thereof in a solvent. However, it is also possible to apply the bacteriophages or a component thereof as a dispersion, in particular a suspension or an emulsion. The bacteriophages, a component thereof, and / or other ingredients of the dispersion can be present undissolved, in particular as a solid as in the case of a suspension, or in a liquid state as in the case of an emulsion.Complete dissolution of all ingredients, especially bacteriophages or any component thereof, is therefore not absolutely necessary, although it may be advantageous depending on the type of application.

[0050] According to one exemplary embodiment, freeze-dried bacteriophages or a component thereof were dissolved or dispersed before application. It is thus possible to freeze-dry bacteriophages or a component thereof, thereby improving their shelf life, and then dissolve or disperse them only immediately before application—for example, directly before application by the user or even by the manufacturer (so to speak, "pre-packaged"). As already mentioned, this improves the shelf life of the bacteriophages or a component thereof.

[0051] According to an exemplary embodiment, the solution or dispersion further contains at least one nutrient for bacteria, in particular for bacteria that may be found in air conditioning systems. Bacteriophages can multiply in metabolically active bacteria and thereby kill them. Therefore, it is usually necessary to supply the bacteria with nutrients. For the purposes of this application, a "nutrient" is understood to mean, in particular, a substance that can serve as food for the host bacteria of the bacteriophages. Suitable nutrients include, among other things, organic substrates as well as mineral macro- and trace elements and buffer components.

[0052] According to an exemplary embodiment, the solution or dispersion further comprises a Ca 2+ -Concentration in the range of 1 to 10 mmol / l, in particular from 1.5 to 5 mmol / l, particularly from 2 to 4 mmol / l, preferably about 2.5 mmol / l. It has been shown that a Ca 2+- Concentration in the above-mentioned range improves the antibacterial efficacy of the bacteriophages or a component thereof. The Ca 2+ - The concentration can be adjusted by adding an appropriate amount of a calcium salt, such as calcium chloride, to the applied liquid.

[0053] According to an exemplary embodiment, the application of bacteriophages or a component thereof comprises spraying a solution or dispersion containing the bacteriophages or a component thereof onto one or more parts of the air conditioning system. This can be done, for example, using a pressure cup gun, optionally with a spray lance, a metering system, or an alternative application system, as is generally known from the prior art.

[0054] According to an exemplary embodiment, one or more parts of the air conditioning system comprise an evaporator and / or a pollen filter housing. These parts of an air conditioning system, particularly a vehicle air conditioning system, are especially susceptible to bacterial colonization and biofilm formation, making it advantageous to apply the bacteriophages, or a component thereof, directly to the target or intended site of action.

[0055] In a further aspect, the present invention relates to the use of bacteriophages or a component thereof for reducing the bacterial load (for removing, killing, or reducing the number of bacteria) on or in an air conditioning system. In particular, the bacteriophages or a component thereof can be used to remove, kill, or reduce the number of (live) bacteria on or in an air conditioning system, such as a vehicle air conditioning system. For further details of this use, reference is made to the above descriptions of the method according to the invention. In yet another aspect, the present invention relates to the use of bacteriophages or a component thereof for preventing the formation of a biofilm or for removing a biofilm on or in an air conditioning system. Preventing the formation of a biofilm is, in particular, a so-called...The "keep c / ean" approach, while the removal of a (pre-existing or already existing) biofilm corresponds in particular to a so-called "dean up" approach. For further details of this use, reference is made to the above descriptions of the method according to the invention.

[0056] In yet another aspect, the present invention relates to the use of bacteriophages or a component thereof to prevent the formation of (bacterially caused, unpleasant) odors in an air conditioning system. Sphingomonas and Methylobacteria bacteriophages have proven particularly suitable for use in preventing the formation of unpleasant odors in an air conditioning system. For further details of this use, reference is made to the above descriptions of the method according to the invention.

[0057] In yet another aspect, the present invention relates to a bacteriophage or a component thereof, comprising at least one from the group consisting of Bacteriophage SP-P_L_l_2022, filed under DSM 34460, and Bacteriophage SP-P_M_l_2022, filed under DSM 34461.

[0058] The present invention is further described by reference to the following examples, which, however, serve only to illustrate the teachings of the invention and are in no way intended to limit the scope of the present invention.

[0059] Examples on a model biofilm

[0060] 1. Examined evaporator plate

[0061] Modern car air conditioning systems typically use evaporator plates made primarily of aluminum. Manufacturers optimize the material properties through additional additives and coatings designed to extend the material's lifespan. Some of these coatings are also intended to have antimicrobial properties. In this case, they would prevent the intended investigations by inhibiting the growth of the bacteria used. Therefore, for this study, metal samples were obtained that were not coated with antimicrobial substances.

[0062] A total of four sheet metal samples, each measuring 20 x 28 cm and with a material thickness of 0.4 mm, were provided. These samples were aluminum evaporator disc strips supplied by MAHLE (Stuttgart).

[0063] The sheet metal pieces were cut into equal-sized sections (e.g., 5 x 5 cm) for testing. To degrease them, the sections were washed with dish soap and then rinsed with ultrapure water until no more foam appeared. The sheet metal pieces were then pre-sterilized (dry heat at 100°C or 140°C).

[0064] 2. Test organisms

[0065] For the investigations, the host bacterium Sphingomonas ssp. SP-H_M_l_2022, registered under DSM 34459, was used. Subcultures were inoculated from a master plate and incubated aerobically for 2-3 days at 30 °C on a horizontal shaker. The densely colonized culture was then adjusted to the required initial bacterial count. The corresponding phage SP-P_M_l_2022, registered under DSM 34461, was taken from a 4 °C chilled container and pre-diluted to the required initial phage count using SM buffer.

[0066] 3. Test variants

[0067] Bacteriophages can only multiply and kill metabolically active bacteria. Therefore, it is necessary to supply the bacteria with nutrients. A high infection rate can be promoted by the presence of phages, growing bacteria, and higher concentrations of calcium carbonate (CaC). Therefore, samples with 0 mM, 2.5 mM, 5 mM, and 10 mM CaC were prepared.

[0068] 3.1 Variant 1: Softagar-coated sheets

[0069] The experimental setup involved distributing the test bacteria (0.1 mL of a suspension with an OD550 nm of 0.5) onto metal sheets in a soft agar (1.2 mL with 4 g agar / L) containing NAR medium and 0–10 mM CaC. After cooling the agar, 250 pL of a phage solution (with approximately 10 million phage units / mL) were added dropwise. The samples were then incubated for several days and evaluated for visually detectable differences. As a control, samples were prepared using sterile SM buffer instead of phage solution. Additionally, samples were prepared where the agar film was deposited not on metal sheets but on plastic culture dishes. Comparing the data for the plastic culture dishes and the metal sheets was intended to reveal whether the metal affected the efficacy of the phages.

[0070] Even distribution of the agar onto the trays was only achieved after the trays were preheated and the dosed volume was doubled. Presumably, prolonged exposure to temperatures around 46 °C impaired the bacterial viability. Even after three days of incubation, bacterial growth remained unexpectedly low, even in samples to which no phages were added. No visual differences were discernible between the samples on metal trays and those on plastic culture dishes.

[0071] As a preliminary step, the soft agar from the initial series with 10 mM CaC was transferred to a suspension medium (10 mL SM buffer) and eluted. Dilutions of the initial eluates with SM buffer were used for viable cell count determinations on NAR agar. The evaluation data are summarized in Table 1. The two assay variants to which phages were added showed significantly lower numbers of colony-forming units. The percentage of underreported results was 95% for the Petri dish variant and 79% for the variant with metal plates.

[0072] [Table 1] Live cell counts of the trials without and with phage dosing

[0073] 3.2 Variant 2: Sheets wetted with nutrient solution

[0074] In the second step, the effect of phages on bacteria colonized the sheet metal surface was investigated. The aim was to determine whether the phages were able to reach the bacteria, a prerequisite for infection. It was important that the phage dilution was not too high to allow for successful interaction with the bacteria. For this purpose, 25 mL of NAR broth were added to eight screw-top glass jars, with a sheet metal tray placed in each jar. A bacterial solution with a defined initial cell count was then prepared from a pre-culture. One mL of this bacterial solution was inoculated into each jar and incubated at 30 °C with gentle shaking (45 rpm) over a weekend. As expected, this resulted in turbidity and bacterial growth on the sheet metal surfaces.

[0075] For treatment approaches with and without phages, solutions of NAR broth and CaC with final concentrations of 0 mM, 2.5 mM, 5 mM, and 10 mM were prepared. In DNA-lobind reaction tubes, 0.1 mL of a previously diluted 1:100 phage high-titer solution was added to a 2 mL aliquot of each of the prepared solutions. This solution should then contain approximately 100 million PFU.

[0076] After incubation for 72 h, the bacteria-colonized trays were removed and excess NAR suspension drained off. After transferring them to sterile screw-top jars, four trays were coated on both sides with 1 mL of the phage-containing solution and left to stand for 10 minutes. Subsequently, all eight trays were incubated in approximately 23 mL of the respective NAR-broth-CaCb mixture (30 °C, 45 rpm).

[0077] At various time points, 200 pL aliquots were aseptically withdrawn, pipetted into a microtiter plate, and the turbidity at 490 nm, which indicates bacterial growth, was monitored using a microtiter reader. In samples without phage addition, a steady increase in turbidity was observed. A decrease in turbidity is expected when phages destroy the bacteria. This was confirmed in the investigations. It was also noticeable that visible lightening of the coating on the metal sheets was observed in samples with phage dosing. After the differences in turbidity between samples with and without phages ceased to increase, the viable colony counts of the samples were determined. For this purpose, after approximately 30 h, the bacterial growth on the sheets was suspended by rinsing with pipettes, and these eluates were pre-diluted and then spread onto NAR plates. This yielded colony count results, which are shown in Table 2.

[0078] [Table 2] Final viable cell counts after treatment without and with phages at different CaCb concentrations

[0079] The evaluation of the viable cell counts at the individual dilution levels and CaCb concentrations showed that even without the addition of CaCb, a reduction in bacterial count was achieved by the phages. A dosage of 2.5 mM CaCb resulted in a 99.86% reduction in viable cell counts by the phages. Further increases in the CaCb concentration did not lead to any further increase in phage efficacy. With 5 mM CaCb, a 98.37% reduction in viable cell counts by the phages was achieved, and with 10 mM CaCb, an 87.67% reduction was achieved. The data demonstrate that the different CaCb concentrations influenced bacterial growth and phage development. Consequently, the 2.5 mM CaCb concentration appears to be ideal for phage activity. The present invention has been described with reference to specific embodiments and examples.However, the invention is not limited to this and various modifications thereof are possible without leaving the scope of the present invention.

Claims

REQUIREMENTS 1. Method for reducing the bacterial load on or in an air conditioning system, the method comprising the following: Applying bacteriophages or a component thereof to one or more parts of the air conditioning system.

2. The method of claim 1, wherein the bacteriophages or a component thereof comprise at least one from the group consisting of Sphingomonas bacteriophages and Methylobacteria bacteriophages.

3. Method according to claim 1 or 2, wherein the bacteriophages or a component thereof from the group comprise at least one from the group consisting of Bacteriophage SP-P_L_l_2022, deposited under DSM 34460, and Bacteriophage SP-P_M_l_2022, deposited under DSM 34461, or a component thereof.

4. A method according to any of the preceding claims, wherein the application of bacteriophages or a component thereof comprises the application of a solution or dispersion containing the bacteriophages or a component thereof.

5. The method of claim 4, wherein freeze-dried bacteriophages or a component thereof were dissolved or dispersed prior to application.

6. The method of claim 4 or 5, wherein the solution or dispersion further contains at least one nutrient for bacteria.

7. A method according to any one of claims 4 to 6, wherein the solution or dispersion further comprises a Ca 2+ -Concentration in the range of 1 to 10 mmol / l.

8. Method according to any of the preceding claims, wherein the application of bacteriophages or a component thereof comprises spraying a solution or dispersion containing the bacteriophages or a component thereof onto one or more parts of the air conditioning system.

9. Method according to one of the preceding claims, wherein one or more parts of the air conditioning system comprise an evaporator and / or a pollen filter housing of the air conditioning system.

10. Use of bacteriophages or a component thereof to reduce the bacterial load on or in an air conditioning system.

11. Use of bacteriophages or a component thereof to prevent the formation of a biofilm or to remove a biofilm on or in an air conditioning system.

12. Use of bacteriophages or a component thereof to prevent the formation of odors in an air conditioning system.

13. Bacteriophage or a component thereof, comprising at least one from the group consisting of Bacteriophage SP-P_L_l_2022, deposited under DSM 34460; and Bacteriophage SP-P_M_l_2022, deposited under DSM 34461, or a component thereof.