Lyophilized test kit for measuring the antibacterial bioactivity of phage preparations against Staphylococcus aureus bacteria

Abstract

Each well in one series received at least 50 μl of lysate of one or a combination of the following phages: MB 401, MB 402, MB 403, MB SA2, MB SA3, MB SA5, i.e., 1×10 9 ~1×10 2 Lyophilized test kit for determining the biological antimicrobial activity of phage lysates based on microtiter plates containing 10-fold dilutions at concentrations of

Description

[Technical field]

[0001] The technical solution relates to a test kit comprising a freeze-dried phage lysate acting against the bacterium Staphylococcus aureus, said kit being directly suitable for a highly sensitive test of the antibacterial biological activity of the phage lysate against the pathogenic bacterium Staphylococcus aureus on the user's premises. [Background technology]

[0002] Bacteria of the genus Staphylococcus are frequent causative agents of human and animal diseases. In recent years, they have become a major problem in human medicine as well as in the veterinary environment. According to the World Health Organization (WHO), 33,000 deaths occur annually in EC countries linked to antibiotic-resistant bacteria. Currently, the WHO estimates that by 2050, more people will die from infections caused by antibiotic-resistant bacteria than from cancer diseases. Within the framework of a solution to the antibiotic crisis, new antibacterial agents must be sought, as well as completely new approaches to the treatment and diagnosis of bacterial causative agents of disease (Duval et al., 2019).

[0003] Besides other possible antibacterial agents, one option is presented by bacteriophages and their use against bacteria. The so-called phage therapy is not a new method. Currently, phage therapy is increasingly entering the heart of doctors and scientific teams in the context of bacterial antibiotic resistance (Lin et al., 2017). The principle of phage therapy was described a very long time ago. However, only the development of modern microbiological and molecular biological methods, describing both phages and pathogenic bacteria, could safely and quickly introduce phage therapy into practice. The approach to the treatment of bacterial infections with phages is diametrically different from common antibiotic treatment. While antibiotics are often broad-spectrum, acting across bacterial genera and species, phages are usually very specific, acting only on a particular bacterial genus, species, and possibly a particular bacterial strain. While antibiotics can be applied without detailed knowledge of the bacterial causative agent of the disease, phage therapy requires a bacterial diagnosis.

[0004] In antibiotic treatment, detailed diagnosis is applied only when strong antibiotics are not effective, and it is necessary to establish whether there are antibiotics to which the bacteria are sensitive. For phages and phage mixtures, said diagnosis is necessary before starting phage therapy. Since the mechanism of action of phages and related bacterial diagnosis are different from general treatment procedures, rapid diagnostic methods need to be adapted to the needs of phage therapy.

[0005] Numerous pharmacopoeial methods for testing the activity of antibacterial agents are already routinely used in microbiology laboratories. These include the diffusion method, in which an antibiotic is released from a disk into the surroundings, eliminating bacterial growth in the presence of the antibiotic. Activity is assessed based on the size of the inhibition zone around the disk. For bacteria that are resistant to the antibiotic, there is no inhibition zone. However, this method does not apply well to bacteriophages, as they are much larger than antibiotics and therefore do not diffuse as quickly into the surroundings.

[0006] Another method is the minimum inhibitory concentration (MIC) where the concentration of antibiotic that inhibits the visible growth of bacteria is determined (Andrews, 2002). Said method is evaluated by a mere visual check and does not require a spectrophotometer. Here, the minimum concentration of antibiotic that inhibits the growth of bacteria is determined. For the test, the concentration of bacteria (CFU) must be clearly defined. Similarly, the minimum bactericidal concentration (MBC) is determined, which is the concentration that prevents the growth of bacteria in the medium. For these methods, procedures are defined by national authorities and used by diagnostic laboratories in hospitals and other institutions. However, there is no officially approved method for determining the antibacterial biological activity of phage lysates, so the test results in individual laboratories can vary considerably. A method that probably approximates the MIC measurement of antibiotics is the Appelman test (Merabishvili et al., 2014), which has been used since 1921, however, it is different from the MIC measurement and therefore the results of both tests are not related to each other. In addition, testing the biological activity of phages is a matter of several specialized laboratories that mainly work with phages. Therefore, the entire process from isolation of the pathogenic bacteria to transportation to a specialized facility and feedback to the patient takes a very long time compared to testing the biological activity directly in the facility where the patient is present.

[0007] There are several methods to determine the activity of bacteriophages against a particular bacterium. It is the plaque method, where individual dilutions of the phage are dropped onto a growing bacterial medium in a double layer agar, and based on the formation of bacteriophage plaques, the concentration of the bacteriophage, i.e. plaque-forming units (PFU), is determined (Anderson et al., 2011). The phage may be dropped onto the culture in the respective dilution or added directly to the agar for the bacterial culture. Thus, based on the number of plaques and the respective dilution, the concentration of viable phage in 1 ml of lysate is calculated. In some cases, the values ​​may vary depending on the culture medium used, the bacteria titrated, etc. This method provides a result that determines the number of viable phages, but only provides partial information about the rate of antibacterial biological activity of the phage against the bacteria. Only comparing the drops of the phage onto more strains makes it possible to determine how active the phage is. However, this method poses high material demands, is prone to errors, and involves more steps that need to be optimized in every facility. Other methods such as qPCR can also be used to determine bacterial numbers, but do not provide information regarding the number of phages that are able to grow on the host bacteria (Anderson et al., 2011).

[0008] Companies, laboratories, and collections of microorganisms often possess very specific phages, and their experts know well the methods to determine bacterial susceptibility. In such cases, the patient or the medical facility must collect the pathogenic bacteria, often resistant ones, and send them for testing. Since a specific phage stored in a single source may not work, the bacteria should be sent appropriately to more locations for testing. This makes a rapid and efficient diagnosis of bacterial susceptibility to phages impossible. With regard to the high dynamics of the development of bacterial resistance to antibiotics, a rapid methodology is needed to determine bacterial susceptibility to other antimicrobial agents (which may be bacteriophages). Such a methodology does not exist in diagnostic laboratories for the time being, and there are no phages available to test in a way that can be used routinely only in diagnostic laboratories, or directly at the place where the pathogenic bacteria are isolated. This can be solved by proper training of staff, or by suitable test kits that can be used anywhere.

[0009] Bacteriophages, like other biological materials, are often not stable for long periods in liquid form. This makes it necessary to look for other options to extend the shelf life, i.e. the usability period, of phage preparations or, as in this case, test kits that contain phages. One option to increase the stability of phages is lyophilization, which generally prevents the degradation of proteins and biological materials, thus extending their stability. Summary of the Invention

[0010] The above mentioned drawbacks are eliminated by the test of the bioefficacy of phages acting against Staphylococcus aureus. Lyophilized phages are contained in microtiter plates at various concentrations. Growth medium, buffer or water is added to the wells together with the lyophilisates in a volume of 50-100 μl, and precisely predefined concentrations of bacteria are added to these concentration series, using a similar procedure described for MIC determination applications, representing the routine of diagnostic laboratories. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] In each numbered series, 10 9 (Well A) to 10 2 A 96-well microtiter plate may be prepared in which phage dilutions from A to H (well H) are present. These concentrations may vary, but always at most one order of magnitude lower. This reduction may be caused by titer loss during lyophilization. However, each batch of kits is tested for a loss in titer, and exact titer values ​​are published for each batch. The test kit also comprises test tubes containing lyophilized bacteria that will be sensitive to the phage(s) contained in the test kit. This bacteria will be prepared and tested in the same way as the clinically tested isolates, to serve as a positive control. Plates containing lyophilized phage lysates may be stored at temperatures between 0 °C and 25 °C. In case of improper storage, activity may be affected by phage degradation.

[0012] The mixture of phage and bacteria is incubated at 37°C for 15-20 hours, and the results are evaluated similarly to the MIC. If the bacteria are sensitive to the phage, the sample will not become turbid until a dilution of -4. If the bacteria are resistant to the phage, all or most of the wells will remain turbid. Undiluted samples often do not become turbid, which is the case at high concentrations (10 9 This is caused by the fact that phages inhibit growth but cannot grow on bacteria (on the order of 10 7 Only if the phage is transparent at concentrations of the order of 0.01% (-3 dilution) can a clear conclusion be drawn that the bacteria are susceptible to the phage. The results of this method can then be compared with the effects of antibiotics, since they were determined using a similar methodology, and can be related to each other.

[0013] The main component of the preparation of the test kit is a phage lysate acting against Staphylococcus aureus, i.e., specifically 1 × 10 9 ~1×10 10Concentrations (titers) of phages MB 401, MB 402, MB 403, MB SA2, MB SA3, MB SA5 (Table 1). Dilute the lysates in 10-fold dilutions as follows and aliquot specific amounts into a row of a microtiter plate:

[0014] [Table 1]

[0015] "%G+C" defines the guanine and cytosine content.

[0016] Preparation of stock lysates

[0017] 1. In a sterile 50 ml test tube, add a minimum concentration of 1 x 10 9 ~1×10 10 Administer 50 ml of phage lysate.

[0018] 2. Aseptically dispense liquid sterile SA medium (tryptone 5 g / l, yeast extract 2 g / l, NaCl 5 g / l) into another seven test tubes (numbered 2 through 8).

[0019] 3. Aseptically transfer 5 ml of the correct volume of dissolution solution from test tube 1 to test tube 2 and thoroughly mix test tube 2 (Figure 1).

[0020] 4. Aseptically transfer the exact volume of 5 ml of lysate from test tube 2 to test tube 3 and thoroughly mix test tube 3. Continue in the same manner up to test tube 8. Finally, test tubes 1-7 contain 45 ml of lysate and test tube 8 contains 50 ml of the most dilute lysate.

[0021] Preparation of plates from stock lysates

[0022] A series of wells in a microtiter plate are always diluted with 100% ethanol in decreasing order from highest to lowest dilution (well A to well H of the plate). 9 ~10 2 containing one phage strain at successive orders of magnitude different dilutions.

[0023] 1.1×10 9 From the 50 ml tube containing phage at a concentration of 100 μl of lysate, transfer 100 μl to well A in the plate. 8 ) lysate is transferred to the next well in series 1.

[0024] 2. Proceed in a similar manner until all wells of series (column) 1 in the microtiter plate are filled (Figure 2).

[0025] 3. In a similar manner, other series (2 to 12) can be prepared in microtiter plates, where 12 different phages can be included or 12 samples of bacteria can be tested (Figure 2).

[0026] 4. The microtiter plates are placed in a freeze-dryer and after freeze-drying is completed, the titers of each row in a randomly selected batch of plates are determined.

[0027] Two strains of S. aureus (susceptible 1137 and less susceptible resistant SA A 4609 FNO) are inoculated into 10 ml of medium and incubated at 37 ° C for 18-24 hours. The inoculum for testing the bioefficacy of the phages is prepared from the culture thus grown. The night culture is diluted to 1 McFarland turbidity value and 2 ml of this dilution are supplemented to 20 ml with medium / physiological solution. This suspension is poured into a sterile Petri dish. The sterile tip of a multichannel pipette is immersed in the suspension and, after immersion, is transferred and moistened to the dissolved lyophilisate in a strip microtube or a microtiter plate. Incubation is carried out at 37 ° C for 12-18 hours, ideally with gentle shaking. The results are evaluated based on the turbidity in the individual wells of the plate (Table 2). The results show clear susceptibility differences in the bacteria S. aureus 1137 and the less susceptible / resistant strain SA A 4609 FNO.

[0028] [Table 2]

[0029] Numbers identify the dilution, K- is a bacterial growth control, KS is a control for sterility in the strip and lysis. Lysis is marked in light grey, moderate turbidity in dark grey, and turbidity in black.

[0030] Patent deposits of the phages are deposited in accordance with the Budapest Treaty at the Munich Collection of Microorganisms (DSMZ) under numbers: S. aureus MB401 = DSM33472, S. aureus bacteriophage MB402 = DSM33473, S. aureus MB403 = DSM33474, S. aureus bacteriophage MBSA2 = DSM33475, S. aureus bacteriophage MBSA3 = DSM33476, S. aureus bacteriophage MBSA5 = DSM33477. [Brief description of the drawings]

[0031] [Figure 1] Example of dilution of phage lysate in one series of wells of a microtiter plate. [Diagram 2] Example of a microtiter plate containing six strains of phages (Table 1) at various dilutions. [Diagram 3] Example of results of the test of bioavailability of phage lysates. In case of equivocal results (possible resistance as well as susceptibility), further testing by titration is appropriate. EXAMPLES

[0032] Example 1

[0033] 100 μl of the appropriate culture medium is pipetted into the wells of a microtiter plate to dissolve the phages. An isolated culture of Staphylococcus aureus is inoculated into 10 ml of medium and grown at 37 °C for 18-24 h. An inoculum for testing the biological activity of the phages is prepared from the grown culture. The overnight culture is diluted to 1 McFarland turbidity value and 2 ml of this dilution is supplemented to 20 ml with medium / physiological solution. This suspension is poured into a sterile Petri dish. The sterile tip of a multichannel pipette is immersed in the suspension and, after immersion, is transferred to the well of the microtiter plate with the dissolved lyophilisate to wet it. The plate is incubated at 37 °C for 12-18 h under moderate shaking, ensuring aeration of the culture. The results are evaluated based on the turbidity of the individual test tubes (Figure 3). To ensure the accuracy of the results, the test of biological activity should be performed in three parallel experiments.

[0034] Example 2

[0035] An isolated culture of Staphylococcus aureus is inoculated into 10 ml of medium and grown at 37 °C for 18-24 h. 100 μl of the appropriate culture medium is pipetted into the wells of a microtiter plate to dissolve the phages. An inoculum for testing the biological activity of the phages is prepared from the grown bacterial culture. 5 μl of night culture is added to 5 ml of medium (or physiological solution). 100 μl of this suspension is transferred to the wells of the microtiter plate containing the dissolved lyophilized phages. The plates are incubated at 37 °C for 12-18 h. The results are evaluated based on the turbidity of the individual wells (Figure 3). To ensure the accuracy of the results, tests of bioefficacy should be performed in three parallel experiments.

[0036] Example 3

[0037] An isolated culture of Staphylococcus aureus is inoculated into 10 ml of medium and grown at 37 ° C to a McFarland turbidity of 0.5. An inoculum for testing the biological activity of the phages is prepared from the grown culture. 2 ml of this dilution is supplemented to 20 ml of medium / physiological solution. This suspension is poured into a sterile Petri dish. The sterile tips of a multichannel pipette are immersed in the suspension and, after immersion, they are transferred and moistened into the wells of a microtiter plate. Before the bacterial culture is prepared, the lyophilisate must be dissolved in the wells of a microtiter plate in 100 μl of an appropriate medium. The plate is incubated at 37 ° C for 12-18 hours. The results are evaluated based on the turbidity in the individual test tubes (Figure 3). To ensure the accuracy of the results, the test of biological activity should be carried out in three parallel experiments. [Industrial Applicability]

[0038] Plates containing the various types of phages (Table 1) are kept in a refrigerator, and if the patient's antibiotic treatment is ineffective, bacterial strains are directly tested for susceptibility to bacteriophages in the facility where the bacteria were isolated and identified.

[0039] References

[0040] Anderson, B., Rashid, MH, Carter, C., Pasternack, G., Rajanna, C., Revazishvili, T., Dean, T., Senecal, A., & Sulakvelidze, A. (2011). Enumeration of bacteriophage particles. Bacteriophage. https: / / doi.org / 10.4161 / bact.1.2.15456

[0041] Andrews, J. M. (2002). Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy. https: / / doi.org / 10.1093 / jac / dkf083

[0042] Duval, R. E., Grare, M., & Demore, B. (2019). Fight against antimicrobial resistance: We always need new antibacterials but for right bacteria. In Molecules. https: / / doi.org / 10.3390 / molecules24173152

[0043] Lin, D. M., Koskella, B., & Lin, H. C. (2017). Phage therapy: An alternative to antibiotics in the age of multi-drug resistance. World Journal of Gastrointestinal Pharmacology and Therapeutics. https: / / doi.org / 10.4292 / wjgpt.v8.i3.162

[0044] Merabishvili, M., Vandenheuvel, D., Kropinski, A. M., Mast, J., De Vos, D., Verbeken, G., Noben, J. P., Lavigne, R., Vaneechoutte, M., & Pirnay, J. P. (2014). Characterization of newly isolated lytic bacteriophages active against Acinetobacter baumannii. PLoS ONE. https: / / doi.org / 10.1371 / journal.pone.0104853

Claims

1. Each well of a series was incubated with at least 50 μl of a suspension of one or a combination of the phages deposited in the Munich Collection of Microorganisms under the numbers DSM 33472, DSM 33473, DSM 33474, DSM 33475, DSM 33476, DSM 33477 at 1 × 10 9 ~1×10 2 A freeze-dried test kit for determining the biological antibacterial activity of phage suspensions based on microtiter plates containing 10-fold dilutions at concentrations of PFU / ml.

2. In addition to the phage, each well of the microtiter plate was incubated with CaCl at a concentration of 0.1 to 1 mM. 2 2. The test kit of claim 1, further comprising:

3. 2. The test kit of claim 1, comprising said phage suspension in ten-fold dilutions at a concentration of 1×10 4 to 9×10 8 PFU / ml.

4. 3. A test kit according to claim 1 or 2, which is stable at temperatures between 0 and 25°C.

5. 3. A test kit according to claim 1 or 2, further comprising a known Staphylococcus aureus bacterium.

6. 1. A method for determining the biological antibacterial activity of a phage suspension using at least one phage from the group consisting of the phages deposited in the Munich Collection of Microorganisms under numbers DSM 33472, DSM 33473, DSM 33474, DSM 33475, DSM 33476, DSM 33477, wherein the susceptibility of the bacteria to be tested can be assessed with the naked eye based on the turbidity.

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