A LYOPHILIZED TEST KIT FOR DETERMINATION OF ANTIMICROBIAL BIOLOGICAL ACTIVITY OF PHAGE PREPARATIONS AGAINST THE BACTERIA STAPHYLOCOCCUS AUREUS

Abstract

A lyophilized test kit for determination of biological antimicrobial effectiveness of a phage lysate on the basis of a microtitration plate wherein individual wells of one series contain at least 50 μl of a lysate of one of the following phages or their combination: MB 401, MB 402, MB 403, MB SA2, MB SA3, MB SA5, namely in the ten-fold dilution at the concentrations of 1×10.sup.9-1×10.sup.2.

Claims

1. A lyophilized Staphylococcus aureus (S. aureus) phage test kit for determination of biological antimicrobial activity of a phage lysate, comprising: a microtitration plate comprising individual wells of one or more series of wells, wherein each series of wells comprises a strain of lyophilized S. aureus phage lysate at a concentration differing by one order from 1×10.sup.9 to 1×10.sup.2, wherein the phage is selected from the group consisting of MB401, MB402, MB403, MBSA2, MBSA3, and MBSA5, deposited at the Leibniz Institute DSMZ, German Collection of Microorganisms and Cell Cultures GmbH under the accession numbers DSM 33472, DSM 33473, DSM 33474, DSM 33475, DSM 33476, and DSM 33477, and combinations thereof.

2. The test kit according to claim 1, further comprising CaCl.sub.2 at concentrations of 0.1-1 mM in the individual wells of the microtitration plate.

3. The test kit according to claim 1, further comprising the phage lysate in unit dilutions within one order at concentrations of 1×10.sup.9-9×10.sup.9, 1×10.sup.8-9×10.sup.8, 1×10.sup.7-9×10.sup.7, 1×10.sup.6-9×10.sup.6, 1×10.sup.5-9×10.sup.5, 1×10.sup.4-9×10.sup.4, 1×10.sup.3-9×10.sup.3, and 1×10.sup.2-9×10.sup.2 PFU/ml.

4. The test kit according to claim 1, which is stable at a temperature of 0-25° C.

5. The test kit according to claim 1, further comprising a lyopholized Staphylococcus aureus bacteria sensitive to the strain of the S. aureus phage.

6. A method of determining biological antimicrobial activity of a Staphylococcus aureus (S. aureus) phage lysate, comprising: a) adding microbial culture media to the individual wells of the one or more series of wells of the microtitration plate of the lyophilized test kit of claim 1 to dissolve the lyophilized phage lysate, b) inoculating the dissolved phage lysate with an isolated test sample of S. aureus bacteria, and c) evaluating the sensitivity of the inoculated test sample of S. aureus bacteria with the naked eye on the basis of the turbidity, wherein a clear test sample inoculated with the S. aureus phage lysate at a concentration on the order of 10.sup.7 indicates sensitivity of the test sample of S. aureus bacteria to the phage and a turbid test sample inoculated with the S. aureus phage lysate at concentrations of 1×10.sup.9 to 1×10.sup.2 indicates resistance of the test sample of S. aureus bacteria to the phage.

7. The test kit according to claim 2, which is stable at a temperature of 0-25° C.

8. The test kit according to claim 2, further comprising a lyopholized Staphylococcus aureus bacteria sensitive to the strain of the S. aureus phage.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 is an example of dilutions of phage lysates in wells of one series of a microtitration plate.

[0012] FIG. 2 is an example of a microtitration plate comprising 6 strains of phages (Table 1) in various dilutions.

[0013] FIG. 3 are examples of results of a test of biological effectiveness of phage lysates. In the case of unclear results (may be resistant as well as sensitive), additional testing by dripping is suitable.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0014] A 96-well microtitration plate can be prepared wherein in each numbered series, there will be from A to H phage dilutions from 10.sup.9 (well A) to 10.sup.2 (well H). These concentrations may vary, but they may always be lower by one order at the most. This decrease may be caused by a titer loss during lyophilization. However, each batch of the kit will be tested for titer decrease and the exact titer value will be declared for each batch. The test kit will also comprise a test tube with lyophilized bacteria that will be sensitive to the phage/phages contained in the test kit. This bacterium will be prepared and tested in the same way as the clinically tested isolate and will serve as a positive control. Plates with lyophilized phage lysates can be stored at a temperature from 0° C. to 25° C. In case of unsuitable storage, the activity may be affected due to phage degradation.

[0015] The mixtures of the phage and bacteria are then cultivated at the temperature of 37° C. for 15 to 20 hours and then the results are evaluated similarly to MIC. If the bacteria are sensitive to the phage, no turbidity of the sample occurs up to the dilution of −4. If the bacteria are resistant to the phage, all or most of the wells will remain turbid. In an undiluted sample, no turbidity often occurs, which is caused by the fact that at a high concentration (on the order of 10.sup.9), the phage inhibits growth although it is not able to proliferate on the bacteria. Only if the sample is clear at a concentration on the order of 10.sup.7 (dilution to −3), a definite conclusion can be drawn that the bacteria are sensitive to the phage. The results of this method can then be compared to the effect of antibiotics as they were determined using a similar methodology and can be related to each other.

[0016] The basis for the preparation of the test kit is a phage lysate acting against the bacteria S. aureus, namely in particular the phages MB401, MB402, MB403, MBSA2, MBSA3, MBSA5 (Table 1) at the concentration (titer) of 1×10.sup.9-1×10.sup.10. The lysate is then diluted as follows in a ten-fold dilution and divided in the particular quantity into one row of the microtitration plate.

TABLE-US-00001 TABLE 1 Bacteriophages used in the test kit for determination of biological effectiveness of phage lysates. “% G + C” defines the contents of guanine and cytosine. Bacteriophage Family % G + C Genome size [kbp] MB401 Myoviridae 30 179 MB402 Myoviridae 30.3 140 MB403 Myoviridae 30.2 142 MBSA2 Podoviridae 29.1 18 MBSA3 Myoviridae 30.3 146 MBSA5 Myoviridae 30.3 146

[0017] Preparation of Stock Lysates:

[0018] 1. Dose 50 ml of the phage lysate at a minimum concentration of 1×10.sup.9-1×10.sup.10 into the sterile 50-ml test tube number 1.

[0019] 2. Aseptically dose 45 ml of liquid sterile SA media (Trypton 5 g/l, Yeast extract 2 g/l, NaCl 5 g/l) into another 7 test tubes (numbers 2-8).

[0020] 3. Aseptically transfer the exact volume of 5 ml of the lysate from the test tube no. 1 to the test tube no. 2 and stir the test tube no. 2 thoroughly (FIG. 1).

[0021] 4. Aseptically transfer the exact volume of 5 ml of the lysate from the test tube no. 2 to the test tube no. 3 and stir the test tube no. 3 thoroughly. Proceed in the same way until the test tube no. 8. Finally, there are 45 ml of the lysate in the test tubes 1-7 and the test tube no. 8 contains 50 ml of the most diluted lysate.

[0022] Preparation of a Plate from the Stock Lysates:

[0023] One series of wells of the microtitration plate always comprise one phage strain at dilutions differing by one order on the consecutive orders of 10.sup.9 to 10.sup.2 in the decreasing order from the highest to the lowest dilution (from well A in the plate to well H).

[0024] 1. From a 50-ml test tube comprising phages at the concentration of 1×10.sup.9, transfer 100 μl of lysate into well A in the plate. Transfer another 100 μl of the lysate of the lower dilution of the ten-fold series (1×10.sup.8) to the next well in the series no. 1.

[0025] 2. Proceed in a similar way until you fill all the wells in the series (column) no. 1 in the microtitration plate (FIG. 2).

[0026] 3. In a similar way, in the microtitration plate, the other series (2-12) can be prepared wherein 12 different phages may be contained or 12 samples of bacteria may be tested (FIG. 2).

[0027] 4. Place the microtitration plate in a freeze-dryer and after the completion of the lyophilization, determine the titer in individual rows in randomly selected plates of one batch.

[0028] Two types of the bacteria (sensitive 1137 and less sensitive—resistant SA A 4609 FNO) S. aureus were inoculated into 10 ml of the media and cultivated at 37° C. for 18-24 hours. An inoculum for testing of biological effectiveness of the phages is prepared from the culture grown this way. The night culture is diluted to the turbidity value of 1 McFarland and 2 ml of this dilution are supplemented to 20 ml with the media/physiological solution. This suspension is poured onto a sterile Petri dish. Sterile tips of a multi-channel pipette are dipped into the suspension and after the dipping, they are transferred and wetted in the micro-tubes of a strip or in the dissolved lyophilizate in the microtitration plate. It is cultivated at 37° C. for 12-18 hours, ideally under gently shaking. The result is then evaluated based on the turbidity in individual wells of the plate (Table 2). The results show a clear difference in sensitivity of the bacteria S. aureus 1137 and the less sensitive/resistant strain SA A 4609 FNO.

TABLE-US-00002 TABLE 2 Testing sensitive and resistant bacteria with dilutions of the phages MB401, MB402, MB403, and MBSA2. The numbers identify dilutions, K- is bacteria growth control and KS is control of sterility of work and lysate in the strips. Lysis is marked L, very moderate turbidity is marked VMT, and turbidity is marked T. Dilutions −9 −8 −7 −6 −5 −4 −3 −2 −1 0 Testing Sensitive Bacteria MB401 K- KS T T T T VMT L L L L L MB402 K- KS T T T T VMT L L L L L MB403 K- KS T T T T VMT L L L L L MBSA2 K- KS T T T T VMT L L L L L Testing Moderately Resistant Bacteria MB401 K- KS T T T T T T T VMT L L MB402 K- KS T T T T T T T VMT L L MB403 K- KS T T T T T T T T L L MBSA2 K- KS T T T T T T T VMT L L

[0029] The strains of S. aureus bacteriophage with working identification MB401, MB402, MB403, MBSA2, MBSA3, and MBSA5 were deposited at the Leibniz Institute DSMZ, German Collection of Microorganisms and Cell Cultures GmbH, InhoffenstraBe 7B, 38124 Braunschweig, Germany on Mar. 19, 2020 under the conditions of the Budapest Treaty and were assigned accession numbers DSM 33472, DSM 33473, DSM 33474, DSM 33475, DSM 33476, and DSM 33477, respectively.

EXAMPLES

Example 1

[0030] 100 μl of suitable culture media is pipetted into wells of a microtitration plate and the phages are left to dissolve. An isolated culture of the bacteria S. aureus is inoculated into 10 ml of the media and grown at 37° C. for 18-24 hours. An inoculum for testing of biological activity of the phages is prepared from the grown culture. The night culture is diluted to the turbidity value of 1 McFarland and 2 ml of this dilution are supplemented to 20 ml of the media/physiological solution. This suspension is poured onto a sterile Petri dish. Sterile tips of a multi-channel pipette are dipped into the suspension and after the dipping, they are transferred and wetted in the wells of a microtitration plate with the dissolved lyophilizate. The plate is cultivated at 37° C. for 12-18 hours under moderate shaking to ensure aeration of the culture. The result is then evaluated based on the turbidity in individual test tubes (FIG. 3). To ensure preciseness of the results, the testing of biological activity should be carried out in 3 parallel experiments.

Example 2

[0031] An isolated culture of the bacteria S. aureus is inoculated into 10 ml of the media and grown at 37° C. for 18-24 hours. 100 μl of suitable culture media is pipetted into wells of a microtitration plate and the phages are left to dissolve. An inoculum for testing of biological activity of the phages is prepared from the grown bacterial culture. 5 μl of the night culture is added to 5 ml of the media (or physiological solution). The volume of 100 μl of this suspension is then transferred into the wells of a microtitration plate comprising dissolved lyophilized phages. The plate is cultivated at 37° C. for 12-18 hours. The result is then evaluated based on the turbidity in individual wells (FIG. 3). To ensure preciseness of the results, the testing of biological effectiveness should be carried out in 3 parallel experiments.

Example 3

[0032] An isolated culture of the bacteria S. aureus is inoculated into 10 ml of the media and grown at 37° C. until the turbidity of 0.5 McFarland. An inoculum for testing of biological activity of the phages is prepared from the grown culture. The volume of 2 ml of this dilution is supplemented to 20 ml of the media/physiological solution. This suspension is poured onto a sterile Petri dish. Sterile tips of a multi-channel pipette are dipped into the suspension and after the dipping, they are transferred and wetted in the wells of the microtitration plate. Before the bacterial culture is prepared, lyophilizate must be dissolved in the wells of the microtitration plate in 100 μl of suitable media. The plate is cultivated at 37° C. for 12-18 hours. The result is then evaluated based on the turbidity in individual test tubes (FIG. 3). To ensure preciseness of results, the testing of biological activity should be carried out in 3 parallel experiments.

INDUSTRIAL APPLICABILITY

[0033] A plate with various types of phages (Table 1) will be stored in a refrigerator and in case an antibiotic treatment of a patient is ineffective, the bacterial strain will be tested for sensitivity to bacteriophages directly in the facility that has isolated and identified the bacteria.

REFERENCES

[0034] Anderson, B., Rashid, M. H., 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. [0035] Andrews, J. M. (2002). Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy. https://doi.org/10.1093/jac/dkf083. [0036] Duval, R. E., Grare, M., & Demoré, B. (2019). Fight against antimicrobial resistance: We always need new antibacterials but for right bacteria. In Molecules. https://doi.org/10.3390/molecules24173152. [0037] 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. [0038] 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.