PHAGE CULTURING DEVICE, METHOD FOR PREPARING PHAGES, AND FILTRATION DEVICE FOR SAME

Abstract

A device and a method for clinically providing a preparation of autologous phages, namely, those that can verifiably be traced back to originating from a very specific person and preferably are also only intended for use in this one specific person includes a phage culturing device which is a fluid line system that is sealed off with respect to the outside environment The phages in the fluid line system obtained after at least one-time culturing are separated from bacteria by way of filtration, and preferably by way of tangential flow filtration. The phages separated by way of filtration are transferred into a collection vessel that is connected to the fluid line system and are preferably removed from the fluid line system, using the collection vessel, as a usable, preferably autologous preparation.

Claims

1. A phage culturing device, comprising a fluid line system that is sealed off with respect to the outside environment, comprising: a. an inlet port configured to have a sample vessel connected thereto in such a way that phages from a sample that is inserted into the sample vessel can be transferred via the inlet port into the fluid line system; b. an outlet port configured to have a withdrawal vessel removably attached thereto so that phages can be withdrawn from the fluid line system via the outlet port; c. a filtration device disposed in a fluid line between the inlet port and the outlet port and comprises a filter membrane configured to allow phages to pass in a direction toward the outlet port, while not allowing bacteria to pass; d. at least one nutrient medium reservoir including a nutrient medium; and e. at least one host bacteria reservoir including host bacteria in lyophilized, viable form and which is closed prior to use and which is configured to be connected to the at least one nutrient medium reservoir by a connecting line that is configured to be opened, the at least one nutrient medium reservoir being disposed upstream from the filtration device in the fluid line system, as viewed in a flow direction from the inlet port to the outlet port so that phages that are transferred into the fluid line system can be replicated at least once by way of culturing upstream from the filtration device in the at least one nutrient medium reservoir including the host bacteria.

2. The device according to claim 1, further comprising a valve disposed in the fluid line system between the inlet port and the nutrient medium reservoir and wherein the sample vessel connected to the inlet port is configured to be at least temporarily brought into fluid connection with the at least one nutrient medium reservoir by way of the valve.

3. The device according to claim 2, wherein the sample vessel and the at least one nutrient medium reservoir are each configured to be of variable volume, the sample vessel being configured as a syringe or as a blood collection tube operating by aspiration or vacuum principle and/or the nutrient medium reservoir being configured as a flexible pouch, so that as a result of a change in volume the nutrient medium that has been mixed with host bacteria can be at least partially delivered from the at least one nutrient medium reservoir into the sample vessel and back into the same or a different one of the at least one nutrient medium reservoir.

4. The device according to claim 3, wherein a plurality of the nutrient medium reservoirs are disposed upstream from the filtration device, as viewed in the flow direction from the inlet port to the outlet port and, in the flow direction, are consecutively connected each via a respective switchable 3-way valve to a connecting line leading to the filtration device in such a way that a change in volume of the sample vessel or of a nutrient medium reservoir allows the nutrient medium to be withdrawn from one of the nutrient medium reservoirs and transferred into a nutrient medium reservoir situated downstream in the flow direction.

5. The device according to claim 4, wherein a respective dedicated host bacteria supply is assigned to each of the plurality of nutrient medium reservoirs and each of the dedicated host bacteria supplies is configured to only be brought in connection with the nutrient medium of the assigned nutrient medium reservoir directly at the assigned nutrient medium reservoir by a closed connecting line which is configured to be opened.

6. The device according to claim 5, further comprising a sterile filter is disposed between two of the nutrient medium reservoirs situated consecutively in the flow direction.

7. The device according to claim 1, wherein the filtration device is configured as a tangential flow filtration device in which the nutrient medium flows tangentially across the filter membrane in a reversing manner on one of two sides of the membrane.

8. A set comprising a plurality of the devices according to claim 1, wherein each of the devices includes a different not antibiotic-resistant host bacteria of a type of an antibiotic-resistant bacterial strain.

9. The device according to claim 7, wherein the tangential flow filtration device comprises first and a second cavity each having a variable volume and being connected to one another by way of at least one channel at least regions of the wall of which are formed by the filter membrane and the at least one channel is configured so that across it nutrient medium can be pumped back and forth between the first and second cavities and can flow tangentially at a first side thereof pointing toward the channel interior and which at a second side thereof facing away from an interior of the channel adjoins a third cavity, an inlet channel opening into the first cavity and an outlet channel opening into the third cavity.

10. The device according to claim 9, wherein the first cavity, the second cavity, the inlet channel and the at least one channel connecting the first and second cavities are integrally formed into a first foil as a bulge protruding from a plane of the first foil and the third cavity and the outlet channel are integrally formed into a second foil as a bulge protruding from a plane of the second foil, the first and second foils being welded or laminated, from opposing sides to surfaces of the filter membrane.

11. The device according to claim 10, wherein each of the two foils is covered by a respective flexible shell which is tightly attached to the respective foil by lamination or welding.

12. A method for preparing phages for destroying a predetermined type of multi-antibiotic-resistant bacteria, wherein the following steps are carried out in the phage culturing device according to claim 1: a. mixing a nutrient medium in the at least one nutrient medium reservoir with not antibiotic-resistant host bacteria from the at least one host bacteria reservoir, the not antibiotic-resistant host bacteria being selected as a type strain of a predetermined type of multi-antibiotic-resistant bacteria; b. culturing, so as to reactivate from a lyophilized state, the not antibiotic-resistant host bacteria in the nutrient medium; c. mixing the nutrient medium, which is mixed with the host bacteria, with autologous phages from the sample in the sample vessel connected to the fluid line system, which the multiresistant bacteria destroy, for which purpose the nutrient medium is temporarily transferred into the sample vessel containing the sample and, after contact with the sample, is returned into the nutrient medium reservoir; d. multiplying the phages transferred from the sample in the nutrient medium reservoir by way of culturing over a predetermined time, whereby lysis of the bacteria of the selected type strain replicates those phages that destroy the predetermined multi-antibiotic-resistant bacteria; e. separating the phages, obtained in the fluid line system following at least one-time culturing, from bacteria by way of filtration; and f. transferring the phages separated by way of filtration into a collection vessel that is connected to the fluid line system, the transferred phages comprising a usable, autologous preparation.

13. The method according to claim 12, wherein the phages are transferred together with the nutrient medium from one of a plurality of the nutrient medium reservoirs via a sterile filter into the nutrient medium of a further nutrient medium reservoir which nutrient medium was previously mixed with host bacteria of the same selected type strain, thereby to repeat the culturing at least once, prior to the filtration by the filtration device.

Description

[0050] An embodiment of the invention will be described in more detail based on the drawing.

[0051] The invention preferably utilizes a two-stage phage enrichment method including downstream sterile filtration and filling, utilizing the illustrated device. The device according to the invention shown in the drawing comprises a system including fluid lines, pouches, and filters.

[0052] The device includes an inlet port 1, which is connected to a nutrient medium reservoir 3 by way of a three-way valve 2 and to which, for example, a so-called Monovette, serving as the sample vessel 4, can be connected, which receives a swab tip of a nasal swab from a patient affected by multiresistant bacteria. The swab tip represents a sample 5 containing phages, which the patient who is to be treated later personally carries inside (for example, as a non-autologous immune system).

[0053] The three-way valve 2 is connected via a line to the nutrient medium reservoir 3, which can, for example, be embodied by a blood bag, here for the preculture of the phages from the swab tip 5. This nutrient medium pouch 3 is, in turn, connected to a host bacteria reservoir 6 including a host bacteria supply, for example in a disposable syringe, via a valve 7, in which lyophilized but viable host bacteria are kept available. These host bacteria were selected as the type strain of the multiresistant microbe from which the patient suffers from whom the nasal swab on the swab sample was taken.

[0054] The host bacteria were, for example, previously procured from a recognized collection and do not have any resistance to antibiotics whatsoever. When the sample vessel 4 is being connected to the three-way valve 2, first the valve 7 between the host bacteria reservoir 6 and the nutrient medium pouch 3 is opened, and the lyophilized host bacteria are reactivated and flushed back into the pouch 3 by the nutrient medium being suctioned in from the pouch 3. After a preferably implemented preculturing duration of several hours at a suitable incubation temperature (for example, 37° C.), the three-way valve 2 is opened, and the activated microbes/host bacteria are drawn into the sample vessel 4 by an increase in the volume of the sample vessel 4.

[0055] There, phages from the nasal swab may adsorb to the reactivated host bacteria when the flow around the swab tip 5 occurs. Subsequent incubation, for example, overnight, gives the phages present in the sample the opportunity to lyse the bacteria culture that is subsequently infected. Only the phages that are compatible with these host bacteria are replicated.

[0056] The nutrient medium is, likewise preferably overnight, suctioned from a further nutrient medium reservoir 8, which is connected to the device of the invention, which is preferably designed as a disposable device, via a valve 9 that is opened for this purpose into a second host bacteria reservoir 10, which is only connected to this pouch 8 and, for example, embodied by a syringe. The lyophilized host bacteria of the same type strain as described above that are kept available in this host bacteria reservoir 10 are reactivated by the nutrient medium and are subsequently injected back into the nutrient medium reservoir 8 designed as a main culture pouch. There, the bacteria can replicate, for example overnight, while the same bacteria in the starter culture are lysed, provided that lytic phages were present in the nasal swab.

[0057] Thereafter, the infected host bacteria culture is drawn up from the nutrient medium reservoir 3 of the starter culture into the sample vessel, for example the Monovette, through the three-way valve 2, and, after the valve has been switched or the starter culture pouch 3 has been pinched off, is filtered through a sterile filter 11, which is likewise preferably connected to the three-way valve 2, by being pressed out of the sample vessel 4. The phages, which have preferably been produced overnight in the starter culture, pass the sterile filter 11 and, through an appropriately opened second three-way valve 12 downstream from the sterile filter 11, reach the main culture in the pouch of the further nutrient medium reservoir 8 which, for example, has replicated overnight.

[0058] The phages that have preferably replicated overnight from the starter culture can now, in the main culture pouch 8, infect the host bacteria of the type strain that is not antibiotic-resistant, which have likewise replicated there, and can be replicated to a much greater extent during the incubation, for example over the course of a second night, by virtue of lysis of the host bacteria.

[0059] After the second three-way valve 12 has been switched, the corresponding lysate, including phages, is then conducted into the tangential flow filter 15, for example by way of gravitation, via a feed line 13 and the inlet channel 14 of the tangential flow filter. There, the lysate fills at least the first cavity 16, which is connected to the second cavity 18 by at least one channel, and preferably multiple channels 17. At least the channel is connected via a filter medium 19, which is to say through the pores of the filter membrane, to a third cavity 20. In the filter 15, a strong tangential flow is generated in the at least one channel 17 above the filtration membrane 19 by alternately pressing in the two flexible, retentate-side cavities 16 and 17. Two enveloping plastic shells 21 and 22 that are sealed on ensure that no host bacteria can exit the device, even in the event that one of the cavities 16, 18 or 20 bursts.

[0060] Due to the filtration principle of tangential flow filtration, efficient separation of the replicated phages from the cell debris, which advantageous arose overnight due to lysis, and the still intact host bacteria is achieved. The permeate is collected in the permeate-side cavity 20 of the tangential flow filter 15 and is subsequently drawn into a preferably provided syringe 25 via the outlet channel 23 through a third three-way valve 24.

[0061] After the third three-way valve 24 has been switched, the permeate is preferably pressed through two sterile filters 26, which are connected in series, downstream from the three-way valve 24 by way of syringe pressure. This phage-containing sterile filtrate is collected in a connected collection vessel, for example a preparation pouch 27, and is separated by the device according to the invention, preferably a disposable device, after the valve 24 has been closed. The preparation pouch 27 is attached to an outlet port of the device so as to be removable for this purpose.

[0062] The inlet port 1 and the outlet port 28 can be formed, for example, by Luer lock connectors.

[0063] The preferably autologous phage preparation that is produced in this first application example can be used as a patient-specific formulation for novel medical therapies, such as the phage therapy.

[0064] In a second application example, phage preparations are produced according to the same principle from environmental samples containing type strains of multiresistant microbes from large-scale livestock farming and are admixed to the animal feed as an animal feedstuff supplement to prevent the spread of multiresistant microbes.

[0065] In a third preferred application example, phage preparations produced according to the same principle are sprayed on infected plantations to combat bacterial plant diseases.

[0066] In a fourth preferred application example, phage preparations produced according to the same principle are sprayed onto commercially available foodstuffs or in foodstuff production facilities to combat spoiling agents and pathogens.