EP2252308A1

EP2252308A1 - Method for producing a mixture of bacteriophages and the use thereof in the therapy of antibiotic-resistant staphylococci

Info

Publication number: EP2252308A1
Application number: EP09722387A
Authority: EP
Inventors: Robert F. MÜLLER
Original assignee: PHYTOLINE GmbH
Current assignee: PHYTOLINE GmbH
Priority date: 2008-03-20
Filing date: 2009-03-13
Publication date: 2010-11-24
Also published as: CN101977616B; MX2010009391A; US8685695B2; AU2009226688A1; BRPI0909330A2; EP2103308A1; CN101977616A; CA2717534A1; WO2009115245A1; NZ587712A; US20110014157A1; JP2011517557A

Abstract

The invention relates to a method for producing a mixture of bacteriophages, wherein at least two different bacteriophage strains have been increased separately, wherein a staphylococcus strain having one strain each of a bacteriophage that can lyse staphylococci is grown in a preculture, and then further reproduced in a main culture, and then purified, and the various strains are then mixed, wherein the mixture of bacteriophages used comprises at least two different serotypes of bacteriophages that lyse the specific bacteria of the species staphylococcus.

Description

A process for the preparation of a mixture of bacteriophages and their use for the therapy of antibiotic-resistant staphylococci

Bacterial infections cause many, sometimes fatal diseases. The development of antibiotics has enabled many of these bacterial infections to be successfully controlled. However, the widespread use of various antibiotics has led to the emergence of bacteria, which are often not only resistant to an antibiotic, but against a variety of antibiotics. Such highly resistant bacterial strains are increasingly leading to serious problems, especially in hospitals, because there can be infected by the medical treatments patients with such strains. Resistant to most commonly used antibiotics, these strains can lead to extremely serious illnesses that are often fatal.

In the context of the present invention, an alternative control of bacteria is used. Just as there are viruses that infect plants and animals and often cause extremely serious diseases, there are also viruses that specifically attack bacteria. These viruses are called bacteriophages. They are usually specific to certain types of bacteria and often bind to those areas of the bacterial cell wall where nutrient uptake (e.g., sugars and the like) usually occurs. After adsorption of the bacteriophages to the bacterial cell wall, the bacteriophages inject their nucleic acid into the bacterium. There, the nucleic acid proliferates and bacteriophage-specific proteins are formed. If enough copies of the bacteriophage nucleic acid and sufficient bacteriophage protein are present in the bacterial cell, it lyses the bacterial cell and the bacteriophages formed are released in large numbers into the environment. Then the infection cycle can start anew.

The use of bacteriophages for the therapy of certain diseases has been known for several decades. Especially in Eastern European countries, this type of control of bacteria has been developed. In WO 2004/052274 is describe a process for the preparation of bacteriophage compositions which can be used in the therapy of bacterial infections, in particular those caused by Pseudomonas aeruginosa. The cultivation of bacteriophages is carried out there in a semi-solid culture medium. WO 97/39111 describes a process for the preparation of bacteriophages with a broad host range and their use in therapy. WO 01/50866 discloses the use of a bacteriophage cocktail to destroy bacteria on various solid surfaces, such as operating rooms or animal husbandry facilities, as well as slaughterhouses.

US 2004/0247569 describes pharmaceutical compositions containing bacteriophages capable of inactivating various antibiotic resistant bacteria such as Enterococcus, Staphylococcus aureus, Pseudomonas and others. WO 2006/047870 describes stabilized bacteriophage compositions adsorbed to a matrix. O'Flaherty et al., Applied and Environmental Microbiology [2005], pp. 1836-1842 describe the anti-Staphylococcus bacteriophage K and its use for the control of antibiotic-resistant hospital staphylococci. WO 2007/148919 describes a bacteriophage specific for Staphylococcus aureus and its use in the control of infections. Capparelli et al., Antimicrobial Agents and Chemotherapy [2007], pp. 2765-2773 describe the bacteriophage M ^Sa and its use against Staphylococcus aureus in the mouse model. Rashel et al., JID [2007], pp. 1237-1247 describe the cloning of a peptidoglycan-degrading enzyme, endolysin, from bacteriophage ΦMR11.

The present invention is a process for producing a mixture of bacteriophages, in which first a Staphylococcus strain with a bacteriophage capable of lysing staphylococci is grown in a preculture and then further propagated in a major culture. Several such bacteriophages are then combined into a mixture.

The mixture of bacteriophages used contains at least two, preferably at least three different serotypes of bacteriophages which specifically lyse bacteria of the species Staphylococcus. Preferably used bacteriophage strains are from the phage classes Myoviridae and Podoviridae. It is an essential aspect of the present invention that several different bacteriophages are used, all of which have the common property that they can lyse staphylococci. In particular, the bacteriophages used must lyze those staphylococci which are involved in human and / or animal pathogenic diseases. Particularly preferably, the bacteriophages used according to the invention lyse those staphylococci, in particular Staphylococcus aureus strains, which are resistant to antibiotics, in particular to methicillin. It is an essential aspect of the present invention that several different bacteriophages are present in the bacteriophage mixture. These may be bacteriophages that specifically lyse Staphylococcus aureus strains, and those bacteriophages may be used that also lyse other bacterial species, but such strains are preferably used only in addition to a mixture of bacteriophages that contain at least two different strains Belong to serotypes.

The bacteriophages used in the method used according to the invention can either be exactly characterized bacteriophages or they can originate from a wild isolate. For this purpose, the bacteriophages are first enriched by common cultivation with Staphylococcenstämmen by conventional methods of bacteriophage isolation starting from a suitable source and then used in the method according to the invention.

For safety reasons, the method according to the invention preferably uses a methicillin-sensitive Staphylococcus aureus strain for propagation of the bacteriophages.

Especially in case of problematic infections in hospitals, methicillin-resistant Staphylococcus aureus strains are frequently encountered, which are often abbreviated as MRSA. According to the invention, however, a methicillin-sensitive Staphylococcus aureus (MSSA) strain is preferably used to propagate the bacteriophage mixture. The preferred strain has since been deposited with the German Collection of Microorganisms under the Budapest Treaty under the accession number DSM 18421.

A particularly important advantage of the Staphylococcus aureus strain used according to the invention for propagating the bacteriophages is that in this strain several of the following virulence factors are absent. These are the following toxin determinants located on transferable elements:

tst: 161/93 sea: 619/93 ss: 62/92 sec: 1229/93 sed: 1634/93 see: FRJ918 eta: 1437/00 eta, etb: 1004/00 lukS-lukF: 3925/02 cna: 2773 / 03 seg: N315 seg and see: 2773/03.

In a preferred embodiment, all of the abovementioned virulence factors are not present in the strain used.

As a result, no virulence factors can be transmitted to the bacteriophages of the propagation strain, and bacteriophage replication is particularly safe in this regard.

Another advantage of the propagation strain used according to the invention is that the properties of the bacteriophages propagated in the strain do not change even after several passages.

For the preparation according to the invention of the bacteriophage mixture, a preculture of the Staphylococcus aureus strain used is grown in the first step. When the bacteria have reached a suitable optical density, the bacteriophage to be propagated is added, wherein in the preculture a ratio of bacteria to bacteriophages of about 10 ⁵ to 10 ² bacteria per 1 phage is set.

The preculture is grown together with the bacteriophages until the lysis of the bacteria begins. Then the preculture containing the bacteriophages becomes one Main culture of staphylococci, wherein preferably such a ratio is set that the bacteria are present in significant excess. Particularly preferred is a range of about 50 to 1000 bacteria per bacteriophage. This excess ensures that all bacteriophages that are in the preculture find a suitable bacterium in which they can multiply.

The ratio of bacteriophages to bacteria is also referred to as the so-called MOI value (multiplicity of infection). In a preferred embodiment of the MOI value in the pre-culture, and the main culture was between 10 ^"6 and 1.

Usually, in the method according to the invention, the main culture is carried out in a fermenter of suitable size. In the fermenters, the individual environmental parameters such as air supply, pH, nutrient addition u.a. be controlled continuously. In a preferred embodiment, the cultivation of the main culture takes place under reduced air supply and furthermore the pH is preferably maintained in a range between 5.8 and 7.8.

After fermentation, the bacteriophages are usually obtained from the fermentation broth by first centrifuging and / or filtering off the fermentation medium so that cell components derived from the lysed bacteria are separated off. The supernatant is preferably filtered through one or more filters, wherein the pore size is selected so that in the first filtration remaining bacteria can not pass the filter. The pore size of these filters is preferably in a range between 0.1 and 1.0 μm, preferably between 0.1 μm and 0.5 μm, more preferably between 0.1 μm and 0.45 μm, and most preferably between 0.1 and 0.2 μm. The bacteriophages can pass this filter size due to their smaller size. However, care must be taken to select those filters on which the bacteriophages do not, if possible, adsorb, since otherwise this could lead to filter blockages and a deterioration in the yield of phages.

In a further step, the phage-containing solution is preferably diafiltered so that the bacteriophages can not pass through the pores and thereby be enriched in the solution. By usual microbiological determination methods, the Concentration of bacteriophages are readily determined. The solution preferably contains a concentration of at least 10 ¹⁰ PFU (plaque-forming units) per ml.

In the context of the present invention, a mixture of at least two or more bacteriophages is used. Such a mixture is preferably prepared by first preparing a solution of each phage to be employed having a defined phage concentration, and then mixing these solutions together in the desired ratio. These bacteriophages can be assigned the same phage classes or even different phage classes. In a particularly preferred embodiment, the bacteriophage mixture consists of two different classes of Myoviridae. The bacteriophage mixture particularly preferably has, on the one hand, the type KP3 bacteriophage and / or the bacteriophage designated MP13.3. In addition, the mixture may also comprise other suitable bacteriophages.

The two preferred bacteriophages were deposited on October 18, 2006 at the German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig. The bacteriophage strain MP13.3 was assigned the accession number DSM 18722 and the bacteriophage strain KP3 the accession number DSM 18723. The deposit was made under the Budapest Treaty.

The bacteriophage mixture is prepared by first multiplying each bacteriophage strain individually in the host bacteria. After purification and titer determination of the respective bacteriophage preparation, the mixture is then prepared, wherein the ratio between the individual bacteriophages can vary in the range between 1:10 to 10: 1. Preference is given to using a mixture which consists of approximately equal parts of the two bacteriophages, the proportion in each case relating to the plaque-forming units. To this mixture also further bacteriophage strains can be added, which were propagated separately.

The phage mixture according to the invention can be further treated accordingly for use as a medicament. Care should be taken during these steps not to inactivate the bacteriophages. Protective substances, such as, for example, thickening substances, for example glycerol or polyethylene glycol, are preferably added. Such additives are preferred when drugs are administered in the form of liquids.

In a further preferred embodiment, the phage mixtures according to the invention have stabilizers, with human serum albumin being particularly preferred.

In a preferred embodiment, the phage mixture according to the invention is used as a medicament in that at least one enzyme which has a synergistic effect on the efficiency of the bacteriophage solution is added to the bacteriophage solution. These may be enzymes that are themselves bactericidal. Thus, it is possible to use lysines, such as, for example, lysozyme, which originate from various sources, that is to say from bacteriophages, from bacteria (endolysins, exolysins) or else other sources. Alternatively, lysozyme can be used, which can be isolated from chicken eggs, for example, and can lyse the bacterial cell wall, especially Gram-positive bacteria. Other enzymes which can also be used advantageously belong to the group of proteases and / or peptidases. An example to be mentioned here would be Papain. Finally, the enzymes may also be nucleases or glucosaminidases which, while not directly bactericidal in themselves, will support the bacteriophage effect.

In a particularly preferred embodiment, the enzyme is the protease serralysin, which is produced from the strain Serratia marcescens. Another preferred enzyme is the nuclease benzonase or the Dornase α. Example 7 discloses the superior property of this combination with a biological matrix (saliva).

If the bacteriophage mixture according to the invention is to be applied in another form, it may also be advantageous to lyophilize the mixture. Again, suitable protective components such as various sugars can be added.

The medicaments according to the invention can be applied in the form of a solution, tincture, ointment or lotion. In a further preferred embodiment, such a drug also contains an antibiotic in addition to the phage mixture, which is preferably effective against Gram-positive bacteria. Background of this combination in that the methicillin-resistant bacteria are to be lysed by the phage mixture according to the invention. At the same time it should be prevented that other bacteria that are not resistant to antibiotics can grow up again. Since antibiotic resistance in staphylococci is often encoded on plasmids, antibiotic-sensitive bacteria could ingest the plasmids from the lysed bacteria. This is prevented by the preferred combination.

The phage mixture of the invention is preferably used for the preparation of a medicament for the treatment of infections caused by antibiotic-resistant Staphylococcus aureus strains. In a preferred embodiment, such infections are those which occur on the skin and / or the mucous membranes. These infections can be treated easily and effectively by the bacteriophage mixtures according to the invention. An advantage of this is that the bacteriophage mixture need not be administered intravenously and / or intramuscularly, but can simply be applied to the wound in the form of a solution, powder, or spray or ointment.

The bacteriophage mixtures according to the invention can be used on the one hand for the treatment of an already existing infection. However, according to another aspect of the invention, the bacteriophage mixtures may also be used to prevent the risk of infection with antibiotic-resistant staphylococcal strains. The bacteriophage mixture is used prophylactically in this case. In a most preferred embodiment, the bacteriophage mixtures of the present invention can be used both therapeutically and prophylactically, in particular to either treat or prevent the risk of lung infection. The lungs are often at an increased risk of infection because the respiratory devices easily allow bacteria, especially antibiotic-resistant bacteria, to enter the lungs.

Another preferred application of the phage mixture according to the invention is the application to the eye. With appropriate infection, the phage mixture is administered in the form of eye drops.

The inventive method is shown schematically in Figure 1. In a preferred embodiment of the method according to the invention, benzonase is added to the solution containing bacteriophages. Benzonase cleaves high molecular weight DNA, mainly derived from the lysed staphylococci, so as not to fear infectivity or possible recombination with fragments of staphylococcal DNA. On the other hand, benzonase has a positive influence on the bacteriophage solution in terms of viscosity. Therefore, the benzonase is preferably added either before microfiltration or after diafiltration. If benzonase is added, it is recommended to perform gel chromatography after purification by ion exchange chromatography. As a result, the remaining remains of the benzonase are separated. The molecular weight of the benzonase is between 30,000 and 40,000 daltons. It is therefore recommended that the column material used for gel chromatography has an exclusion volume of about 100,000 daltons, preferably about 50,000 daltons.

An essential further advantage of the benzonase addition is that the yield in the ion exchange chromatography is increased. So less bacteriophages are lost because the complexation with the nucleic acids is positively influenced in terms of a higher yield.

Another preferred aspect of the present invention is that the bacteriophage suspension can be stabilized by the addition of human serum albumin. It may be human serum albumin derived from human plasma. However, any viral contamination must have been removed. Alternatively, recombinant human serum albumin can be used to avoid the risk of viral contamination.

In another preferred embodiment, bacteriophage solution is stabilized by adding magnesium ions at a concentration of between 0.1 and 100 mM, preferably between 1 and 10 mM Mg ²⁺ . In a particularly preferred embodiment, the stabilization is achieved by the addition of a combination of magnesium ions and human serum albumin.

The following examples are intended to illustrate the present invention. Example 1: Phage Production in the Fermenter

1.1 microorganisms

Bacteria for MRSA M.03.02.0028, frozen culture of a strain obtained from fermentation Prof. Daschner, Freiburg. The tribe was at the

DSM deposited under No. 18421 under the Budapest Treaty.

Bacteria for Staphylococcus aureus M.03.02.0008 obtained from Prof. Daschner, Plaquetest Freiburg. The strain was deposited with the DSM under the Budapest Treaty under No. DSM 18421.

Phages mother phages I, sterile filtered

Concentration: 1x10 ¹⁰ PFU / ml

In each case separately the two bacteriophage strains KP3

(DSM 18723) and MP13.3 (DSM 18722).

1.2 culture media

a) APS-LB Broth

20 g of ready medium (Becton / Dickinson, Lot No. 430510) are weighed in, dissolved in deionized water and made up to 1 l final volume. The natural pH of the medium thus produced is about 6.8 and does not need to be adjusted. The medium is sterilized at 121 ⁰ C for 20 min and then stored at room temperature (RT, 20-22 ⁰ C).

b) APS-LB broth (fermentation)

20 g of finished medium (see above) + 2.5 g MgSO ₄ (Roth, pa) are weighed in, dissolved in deionized water and made up to 1 l final volume. The medium is sterilized at 121 ^° C. for 20 minutes in the medium paste and then filled directly into the sterilized culture vessel. c) LB medium

10 g tryptone from casein (Roth, for microbiology), 5 g NaCl (Roth, p.a.) and yeast extract (Roth, for bacteriology) are dissolved in deionized water and made up to 1 l final volume.

For the preparation of solid nutrient medium, 15 g agar agar / l (Roth, high purity) are added. The medium is sterilized at 121 ⁰ C for 20 min. The agar plates are stored at RT. For the test plates were used, which were stored for a maximum of 1 week under these conditions.

d) Bird Johnson agar

58 g of finished medium (Merck) was dissolved in 1 liter of warm deionized water, then sterilized at 121 ⁰ C for 20 min. The medium was cooled to about 50 ^° C., then 0.24 g / l potassium tellurite (sterile filtered solution) was added. The medium is filled in Petri dishes, dried for 30 minutes under the Cleanbench and then stored at 4 ° C.

e) LB top agar

1, 0 g tryptone from casein

0.5 g NaCl

0.8 g agar agar are dissolved in deionized water, made up to 100 ml and then sterilized at 121 ⁰ C for 20 min. The storage until use takes place in

Drying cabinet at 60 ^° C.

1.3 Buffers

a) 0.85% NaCl

0.85 g of NaCl was dissolved in deionized water and made up to 100 ml final volume. The buffer is sterilized at 121 ⁰ C for 20 min.

b) phage buffer

20 mM Tris (pH 7.4), (Roth, Ultra quality)

10 mM NaCl

10mM MgSO ₄ x 7H ₂ O The preparation was carried out from sterile stock solutions and sterile, deionized water. Storage took place at RT.

c) 1M NaOH

40.01 g of NaOH (Roth) were dissolved in deionized water and made up to 1 l final volume. The solution was sterilized at 121 ⁰ C for 20 min, then stored at RT.

d) pH Calibrations pH 4.01 (Mettler Toledo) pH 7.00 (Mettler Toledo)

e) Oxygen probe (pO ₂ probe, measures the oxygen partial pressure of a medium) To calibrate the pO ₂ probe, first select the Calibration button in the task bar, then the pO ₂ button in the next window. Here, too, the temperature value is set to room temperature or the temperature of the solutions is entered.

f) OD probe

OD probe is not calibrated, but only the zero point for each run reset for the respective medium. Since the probe has not yet been linearized, the external comparison with the OD ₆ oo measured at the photometer is essential.

g) pH measurement

The pH meter is switched on before the measurement and the required temperature is set. The pH meter is examined for its calibration state and - if necessary - calibrated according to manufacturer's instructions. The probe is removed from the 3 M KCl solution and rinsed with deionized water. The probe is dried with a paper towel and kept in the solution to be measured. The measuring process can be accelerated by panning or stirring the solution. The measured value is noted, the probe is removed from the solution and rinsed thoroughly with 70% ethanol, then with deionized water, dried with a paper towel and then returned to the 3M KCl solution for storage. 1.5 Cultivation Bacterial strain

of said strain in 1.1 a of scale for the fermentations of glycerol frozen culture was removed from the -80 ⁰ C freezer and thawed under the Clean Bench

2 inoculations of cell material were inoculated into 20 ml of APS LB medium and incubated shaking at 37 ° C and 200 rpm overnight.

1.6 fermentation

the culture was taken from the 37 ° C shaking incubator 0.1 ml was taken for OD measurement, 1 + 9 diluted with medium (0.9 ml APS-LB + 0.1 ml culture) and an OD _60O mean of _1.363 determined via the sampling hose, a sample of the medium was removed and filled into an Eppi (= reaction vessel from Eppendorf). Of these, 500 .mu.l were plated on LB agar and Vogel Johnson agar and incubated at 37 ⁰ C overnight.

1.7 Approximation of overnight culture to OD 8.0

the culture was mixed with medium in a sterile Falcon tube and an optical density of about 8.0 was established by dilution with sterile medium.

1.8 Incubation bacteria / phages

the phages (1.1) to be used were diluted to 2x10 ² in phage buffer in Eppis (final volume: 2 ml of 2x10 ² dilution) when the temperature in the vial was stable, 15 ml were set

Bacterial culture and 2 ml phage mixed in Falconr tube and 20 min at

37 ⁰ C incubated then the bacteria / phage mixture via a cannula into a

20 ml syringe sucked and injected via the septum in the lid of the culture vessel after stirring for 3 min, the first measured values of the fermentation were determined. 1.9 CFU / mL determination

a decadal dilution series of the culture adjusted to OD 8.0 was pipetted according to the following scheme:

Table 1

2x100 μl of the dilution steps 10'-IO ^'8 were plated out on LB agar

(Drigalski smear) the plates were used ü.N. incubated at 37 ° C the next day

1.10 fermentation

After fermentation start, the parameters were read or measured every 30-60 min. the OD ₆ oo measurements were made against the fermentation medium as a blank. Sampling (8 ml each) was performed during the fermentation

Sampling hose each by means of a sterile 10 ml syringe from the 8 ml of 1 ml for OD6oo measurement was taken _ex ternal (either directly added to a cuvette or filled into Eppi), the rest was used for pH

Measurement _e χtern used. Table 2: Values obtained during fermentation

* Due to turbulence due to stirring at 400 rpm, the values fluctuated back and forth, so that the value shown here can only indicate one tendency

X = no measurement performed

recal = values recalibrated (call up via "Calibration" on the fermenter surface, then "Recalibration", enter value) 1.11 results

An overnight culture was used which, after OD adjustment to 8.0, had a concentration of 8.5x10 ⁸ CFU / ml (CFU = Colony Forming Unit).

Calculation MOI value:

Bacteria: 15 ml x 8,5x10 ⁸ CFU = 1, 3x10 ¹⁰ bacteria per ml

Phage: 2 ml x 5x10 ⁷ PFU / ml (by 2x10 ^"2 dilution) = 1x10 ⁸ PFU

(Plaque forming units per ml) MOI: 1/130 (phage / bacteria) = 0.0077

The lysis started this time after a little less than 3 hours. This seems to be the expected lysis onset, as the fermentation was repeated at the same time. The course of fermentation seems to be very reproducible, because the oxygen partial pressure had fallen to 0.0% after every 2 h 15 min and again to> 0.0% 30 min later. Also, the OD measured at certain time intervals was nearly identical over the course. The bacteria each achieved an OD of 1, 5 as their highest value. When the fermentation was stopped after 7 h 15 min, an OD ₆ oo of 0.179 (A12: OD ₆ oo 0.22) was still measurable. However, the high number of viable staphylococci on the Vogel-Johnson agar (see item 5.1.2) in the fermentation product indicated that the fermentation / lysis was not yet 100% complete.

The phage concentration of 5.4x10 ⁹ PFU / ml determined in the fermentation is between the values obtained in other fermentations (3.3x10 ⁹ PFU / ml and 8.3x10 ⁹ PFU / ml). A yield of about 5x10 ⁹ PFU / ml was achieved in the shake flask, so that this result is quite satisfactory. If the lysis continues to progress at 4 ° C, a higher yield can be achieved.

Example 2 - Preparation of a bacteriophage mixture

First, the production strain, a methicillin-sensitive strain of Staphylococcus aureus, which was deposited with the DSM under the number 18421, cultured overnight in about 250 ml of nutrient medium at a temperature of 30 ^c C. By dilution of the grown bacterial culture broth with fresh, sterile medium was an optical Density of about 1, 0 set. The diluted bacterial culture was then spiked with an appropriate amount of the bacteriophage such that a phage to bacterial ratio of about 1 was established. Subsequently, the mixture of bacteria and bacteriophages was preincubated for about 5 hours at a temperature of 30 ° C.

After preincubation for five hours, the bacterium / bacteriophage mixture was transferred to a 10 liter fermentor in which the above-mentioned methicillin-sensitive Staphylococcus aureus strain was grown to an optical density of 2.0 at 37 ^° C. The preferred bacteria mixed with the preculture were fermented for an additional 12 hours at 37 ° C. Thereafter, the fermentation was stopped and the fermentation broth containing bacteria, bacterial residues and phages was first prepurified in a depth filtration. In depth filtration, the fermentation broth is filtered through a filter element having a pore size that readily allows passage of the bacteriophages. Since bacteriophages often have negative charges, the filter element has negative charges to prevent unwanted adsorption of the bacteriophages to the filter element.

After depth filtration, the fermentation broth is filtered through a filter having a pore size of about 0.1 to 1 micron in diameter to remove bacteria and bacterial components, particularly membrane constituents.

Since the solution obtained after the microfiltration containing the bacteriophages is very voluminous, the volume was significantly reduced by ultrafiltration. In ultrafiltration, the pore size of the filter is chosen so that only the solvent of the fermentation broth, ie water and dissolved therein components of the nutrient medium are removed by filtering. As a result, the concentration of bacteriophages is increased significantly, usually a concentration of bacteriophages is achieved by a factor between 100 to 10,000.

The concentrated bacteriophage mixture was then spiked with benzonase, removing the remaining bacterial nucleic acids. Subsequently, the bacteriophage solution was adsorbed to an ion exchanger and then eluted with a degree of salinity. The fractions containing the bacteriophages were then further purified by gel chromatography and the resulting bacteriophage solution was adjusted to the desired concentration of the bacteriophages set. For the preparation of the mixture, the two preferred bacteriophages according to the invention were separately grown and purified and then mixed.

The bacteriophage mixture was then brought into a desired shape, that is a coatable solution, ointment, powder or granules, filled and can be used.

Example 3 - Stability of the bacteriophage preparation at 4 ° C, room temperature and at 37 ° C

A phage buffer with the following composition was used as the storage buffer: 20 mM Tris (pH 7.4), 100 mM NaCl, 10 mM MgSO ₄ .7H ₂ O. The preparation was carried out from sterile stock solutions and sterile deionized water. The stabilizer used was human serum albumin (HSA) at a concentration of 1 g / l. The product was purchased from Baxter.

The two bacteriophages MP13.3 and KP3 were investigated.

For the determination of the PFU Staphylococcus aureus, deposited with the DSM under the number 18421 in LB medium was grown. With the overnight culture, a 100 ml Erlenmeyer flask was inoculated with 20 ml of LB medium so that an OD _60O of 0.2 was achieved. The culture is then incubated at 37 ° C. and 200 rpm in a shaking incubator until an OD ₆₀₀ between 0.8 and 1 is reached.

From the phage solution to be examined, a decadic series of dilutions is applied in phage buffer (100 μl phage + 900 μl phage buffer).

100 μl of the diluted phages are pipetted in duplicate with a sterile filter tip onto the bottom of sterile, labeled centrifuge tubes. Into a tube, 100 μl of phage buffer are added as a control. Then all tubes are mixed with 100 μl DSM 18421 (OD ₆₀₀ 0.8 to 1.0). The tubes are sealed with aluminum caps, gently shaken and pre-incubated for 20 min at 37 ⁰ C together with the corresponding agar plates in an incubator. About 2 minutes before the pre-incubation, LB top agar is removed from the 60 ° C cabinet and mixed for 1-2 minutes at room temperature on a magnetic stirrer. The warmed plates, tubes and top agar are placed under the cleanbench after incubation.

6 ml of Topagar are drawn up with a 5 ml pipette (exact scaling above 5 ml) and distributed on two centrifuge tubes of 3 ml each. The tubes were mixed for 2 seconds on the vortexer and then poured out quickly on the associated agar plates and distributed. The agar plates are dried under the cleanbench for at least 10 minutes before being transported to the 37 ° C incubator and incubated "upside-down" overnight.

Stability of MP4 with and without HSA values as PFU / ml

Table 3

Table 3 shows the results of stabilization, which could be achieved by the addition of human serum albumin using the bacteriophage MP13.3 as an example. The notation used in Table 3 is another representation of the phage concentration. Such means, for example, 3.10 E + 09: 3,10 ^'10 ⁹ PFU phage per ml of solution.

The long-term stability was also investigated using the example of MP4, where the preparations were measured at 4 ° C. over a period of up to 32 weeks. The results are shown in Table 4. The abbreviation M-phage (MP) stands for the bacteriophage MP13.3 (DSM 18722) and the abbreviation K-phage stands for the bacteriophage KP3 (DSM 18723). Store at 4 ° C with 1 mg / ml HSA (Baxter) values as PFU / ml

Table 4

Example 4 - Determination of specific activity

a) Determination of protein concentration

Coomassie Blue G-250 forms a blue color complex in an acidic environment with protein, which can be photometrically quantified at λ = 595 nm using a microtiter reader (96 wells / microtiter plate, 300 μl / well).

Materials:

Coomassie® Protein Assay Reagent (Ready-to-use) from Pierce (Order Number: 23200)

Standards (500 μl aliquots at 1 mg / ml): o γ-globulin (e.g., BioRad, Cat. No .: 500-0005); Bovine serum albumin (BSA) (e.g., BioRad, Order No .: 500-0007);

Method:

An aliquot of the adequately diluted sample was added to the reagent and photometrically measured within a defined time interval. The total protein concentration in mg / l or μg / l was calculated from the standard curve taking into account the dilution factor. b) phage activity

Test principle:

The quantitative determination of the infectivity / activity of a phage suspension is carried out by the plaque test. The principle is based on the specific for Staphylococcus aureus lytic effect of the phage used. The proliferation of phages within a host cell lawn creates clear, macroscopically visible foci, so-called "plaques", which are clearly separated from the environment.

Exactly one plaque, assuming an appropriate MOI (multiplicity of infection), can be attributed to the infection of a bacterial cell with a single infectious phage. The phage activity can be calculated in PFU / ml (Plaque Forming Unit) or PBE / ml (Plaque Forming Unit) on the basis of the sample volume used, the sample dilution and the determined number of plaques per plate.

If the phage titer of the sample to be measured is known, the dilution and the sample volume are chosen such that the number of plaques per agar plate is between 50 and 250. If the phage titer of the sample is not well known, a wider range of sample volume or dilution levels should be covered.

Materials:

Staphylococcus aureus DSM 18421.

Method:

Cultivation of Staphylococcus aureus in a shake culture in two stages.

Short-term incubation of the sample aliquot with an aliquot of the Staphylococcus culture.

Mix the suspension with liquid agar and plating the agar on a prepared agar test plate.

Incubation of the agar test plate for 18 - 22 h.

Counting of plaques.

Calculation of activity in PFU / ml. c) Specific activity

Specific activity is defined as the ratio of activity to protein in PFU / μg protein. The following values were measured:

Table 5

In the solution used, the phages used according to the invention preferably have a specific activity of at least 10 ⁸ PFU / μg protein and particularly preferably a specific activity of 9 10 ⁸ (corresponds to 0.09E + 10) for the K phage and 2.4 10 ⁹ ( 0.24E + 10) for the M phage (each PFU / μg protein). For different batches, the following values were determined:

Table 6

Example 5 - Use of Benzonase

Benzonase is an endonuclease that degrades DNA as well as RNA with high efficiency to the smallest units. This enzyme is mainly used to reduce the viscosity. Another field of application is above all the prevention of the interaction between DNA and large proteins. This interaction can make efficient purification difficult or even impossible. This applies in particular to the present process, in which the very large protein shell of the phage has to be taken into account. The inventive method was carried out once without benzonase and once with benzonase treatment. The results are shown in Tables 7 and 8.

Table 7: Ion exchange chromatography without benzonase

Table 8: Ion Exchange Chromatography with Benzonase

Example 6 - Effectiveness of the mixture

To prove that the mixture according to the invention of bacteriophages has surprising properties, various pathogens from a clinic were tested. The staphylococcus aureus strains used with multiple antibiotic resistance were isolates provided by a clinic. It was tested whether the different Staphylococcus aureus isolate could be lysed once by the MP13.4 phage, once by the phage KP4 and once by a 1: 1 mixture of MP13.4 and KP4.

Table 9 below shows that the use of a phage mixture also lyses Staphylococcus aureus strains that can not be lysed by a single bacteriophage. Since in the clinic in the therapy of multiple antibiotic-resistant strains of staphylococci, these pathogens must be lysed as quickly and effectively as possible, it is of invaluable advantage if a bacteriophage mixture can lyse as many different problem germs and thus destroy them.

The strains are problematic isolates from a clinic that are resistant to a variety of antibiotics.

Table 9: Comparison of bacteriophage efficacy, both individually and as a mixture in resistant Staphylococcus aureus strains

0 means no lysis; + means weak lysis, ++ means better lysis and +++ means very good lysis Example 7 - Lysis of Staphylococcus aureus DSM 18421 in saliva

7.1 Preparation of a saliva mixture

Morning saliva is collected individually from various persons (before breakfast and before brushing). The saliva samples are centrifuged for 5 min at room temperature at 12,000 rpm. The supernatant is removed and sterile filtered (0.8 μm prefilter, then

0.22 μm) the saliva samples are mixed 1: 1 in a sterile container.

7.2 Incubation of the lysis mixtures

in the prepared saliva prepared as described above are also added: approx. 1x10 ⁴ CFU / ml DSM 18421 (from appropriate dilution of the overnight culture) approx. 1x10 ⁸ PFU / ml phage (Simba Top, 1: 1 mixture of purified M and K -

Phages) additionally are serralysin (100 U / ml), Dornase (25 U / ml) or benzonase

(25 U / ml) or a combination of the enzymes used the enzymes are added right at the start of the incubation a growth control is carried out in which the phages are replaced by adding the same volume of storage buffers (composition see below) all the mixtures contained in the sum Incubate 2 ml in 10 ml penicillin glass bottles at 37 ° C and 200 rpm

(Incubation Shaker) performed.

7.3 Determination of the number of live cells (CFU / ml) after 6 h

After 6 h incubation period, an aliquot is taken from each batch and decadently diluted in storage buffer from each dilution series 2-5 suitable dilution stages are selected and each 2x100 .mu.l (double batch) of which plated (by means

Drigalski spatula) for the phage-free approaches, on LB agar, for phage-on-LB agar approaches.

Agar with 0.01% citrate plated after at least 16 h incubation at 37 ^° C., the colony forming units (CFU) are counted on each plate for the calculation of the CFU / ml, only plates are used which are at least 20

CFU have the graph done by the CFU / ml plot

(logarithmized) over the incubation period.

The results of the experiment are summarized in Tables 10 to 12.

Table 10: Serralysin with and without phages:

Table 10 (Serralysin with and without bacteriophages) describes, by way of example, the effect of serralysin on the concentration of S. aureus in saliva alone and in combination with bacteriophages (mixture of DSM 18722 and DSM 18723). As the table shows, serralysin alone does not result in a reduction in colony count. The bacteriophage mixture shows a reduction in colony count to 67.4 percent. In the combination of bacteriophages and serralysin, the colony count drops to 18.7 percent. Thus, a synergistic effect is given.

Table 11: Comparison of Dornase α / Benzonase

Table 11 (Comparison of Dornase α / Benzonase) describes an example of the influence of the nucleases Dornase α and Benzonase on the concentration of S. aureus in saliva. The use of bacteriophages leads to a reduction of the colony count to 18.7 percent. The combination of bacteriophage and nuclease reduced the colony count to 8.88 percent for Dornase α and 6.37 percent for Benzonase.

Table 12: Comparison of Dornase α / serralysin combination with single enzymes

Incubation period: 6 hrs.

Medium: saliva (human; mixture)

Table 12 (Comparison of the single-enzyme combination of Dornase α / serralysin) describes, by way of example, the comparison of the effect of the nuclease Dornase α and the proteinase Serralysin individually and in combination on the synergistic effect with bacteriophages. The enzymes alone reduce the colony count of S. aureus in saliva to 54.3 percent (Dornase α) and to 28.2 percent (Serralysin). The combination of both enzymes leads to a further reduction of the colony count to 20.0 percent.

Example 8: Detection of the Effect of a Combination of the bacteriophages DSM 18722 and DSM 18723 on a Lethal Staphylococcus aureus Infection of the Lung in the Immunosuppressed Mouse

Comparison with the use of antibiotics or comparison of different application times of bacteriophages

The in vivo efficacy of a mixture of bacteriophages DSM 18722 and DSM 18723 was demonstrated in an animal model (immunosuppressed mouse, intratracheal application of a virulent S. aureus strain). Also, for comparison a non-sensitive (ciprofloxacin) and a sensitive antibiotic (vancomycin) used.

Four groups of 20 mice each weighing approximately 25 g were first subjected to immunosuppression to ensure sufficient infection of the mice with S. aureus. Immunosuppression started with cyclophosphamide 4 days before the bacterial infection. One day before, another cyclophosphamide application was performed, along with a single dose of cortisone. Immunosuppression with cyclophosphamide was repeated 3 days apart.

As immunosuppression may possibly lead to an undesired, lethal influence of existing pathogenic germs in the mice (as could be shown in preliminary experiments), the animals were shielded with the antibiotic ciprofloxacin over the period of the experiment. The S. aureus strain selected for the infection is resistant to this antibiotic.

The S. aureus strain selected for the infection (internal name M.06.02.0071) is an isolate provided by the Robert Koch Institute in Wernigerode. S.aureus belongs to the group of USA300 (MRSA, ST8) strains which are considered to be particularly virulent due to the presence of the virulence factor PVL. The selected strain is u.a. resistant to ciprofloxacin but sensitive to vancomycin and clindamycin as well as some other antibiotics.

On day 0, the mice were administered intratracheally 1, 5 × 10 ⁷ CFU of S. aureus under anesthesia.

As a medication, either 5.5 × 10 ¹⁰ PFU of a 1: 1 mixture of the bacteriophages DSM 18722 and DSM 18723 were administered intratracheally under anesthesia one hour before the infection or one hour after the infection. Furthermore, one group was treated subcutaneously with vancomycin twice daily starting one day prior to infection.

The main parameter was the determination of lethality as a function of the duration of the test. The trial duration was 14 days. The result of the experiment is shown in Table 13:

Percent surviving animals

Table 13

There is a clear effect of bacteriophages on lethality. In particular, administration of the bacteriophages prior to infection results in a significant reduction in mortality in the acute course. The influence of bacteriophage administration after infection is also evident. With the previous and then continued use of the antibiotic vancomycin, a reduction in mortality is also achieved in the acute phase compared to placebo, albeit slightly lower than with the one-time administration of the bacteriophages prior to infection.

The protective effect of the single administration of the bacteriophages prior to the infection is more pronounced at the end of the experiment (after 14 days) than with the protective or continuous administration of the antibiotic vancomycin.

Compared to the protective and then continuous administration of the non-sensitive antibiotic ciprofloxacin, the effect of bacteriophages as an option in the treatment of S. aureus infections becomes particularly clear. Example 9: Nano calorimetric measurements

For the investigation of the activity of microorganisms, another sensitive method is available with nanocalorimetry. The metabolic rate is measured by the heat production. The heat production is determined by the voltage at thermocouples (in [μV]). Activators and inhibitors of metabolism influence the heat production and thus the voltage at the thermocouples.

A detailed description of the measuring system used and the procedure can be found under: J. Lerchner et al. Thermochimica Acta 477, 2008, 48-53.

As part of the investigations on the effectiveness of bacteriophages, the following nanocalorimetric investigations were carried out:

- Influencing the metabolism of S.aureus DSM 18421 by a 1: 1 mixture of bacteriophages DSM 18722 and DSM 18723

- Influencing the metabolism of S.aureus DSM 18421 by the antibiotics vancomycin and clindamycin.

Influencing the metabolism of S.aureus M.06.02.0071 by a 1: 1 mixture of bacteriophages DSM 18722 and DSM 18723 Influencing the metabolism of S.aureus M.06.02.0071 by the antibiotics vancomycin and clindamycin.

- Influencing the metabolism of S.aureus M.06.02.0071 by a 1: 1 mixture of bacteriophages DSM 18722 and DSM 18723 or by the antibiotics vancomycin and clindamycin after they were used in the mouse pneumonia model and isolated from lung tissue.

Both the S.aureus strain DSM 18421 and the strain M.06.02.0071 are sensitive to the individual bacteriophages DSM 18722 and DSM 18723 and to the antibiotics vancomycin and clindamycin.

To carry out the experiments, vancomycin and clindamycin were each added at a concentration of 10 μg / ml (corresponding to more than 10 times the MIC present in Preliminary tests was determined). The bacteriophages had a concentration of

4 x 10 ⁹ PFU / ml.

The results of the measurements are shown in Tables 14-16. It shows that the antibiotics in the bacteria lead to a residual activity, which is interpreted as an indication of a stasis. The bacteria grow again after falling below the minimum inhibitory concentration (MIC). This creates a risk for the development of resistance or inadequate therapy.

By contrast, the action of the bacteriophages practically leads to irreversible inactivation by the lysis of the bacterium and thus prevents the possibility of renewed growth. Low residual heat production is attributed to enzymatic processes that cause degradation of the biological material.

The nano calorimetric measurements show that the effect of the bacteriophages is superior to that of the antibiotics used.

S.aureus DSM 18421

Table 14 S.aureus M.06.02.0071

Table 15

S.aureus from tissue (M.06.02.0071, mouse pneumonia model)

Table 16

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Claims

Claims:

A process for the preparation of a mixture of bacteriophages, wherein at least two different bacteriophage strains have been propagated separately, wherein a strain of Staphylococcus each with a strain of a bacteriophage capable of lysing staphylococci is grown in a preculture and then further propagated in a major culture and subsequently is purified and the various strains are subsequently mixed, characterized in that the mixture of bacteriophages used contains at least two different serotypes of bacteriophages which specifically lyse bacteria of the species Staphylococcus and that the bacterial strain used to propagate the bacteriophages comprises a methicillin-sensitive Staphylococcus aureus Trunk is.

2. The method according to claim 1, characterized in that the methicillin-sensitive Staphylococcus aureus strain has the accession number DSM 18421.

3. The method according to any one of the preceding claims, characterized in that the precultivated mixture of bacteria and bacteriophages is added to a preferred culture of staphylococci, wherein the ratio of phage to bacteria is adjusted so that the MOI value between 0, 01 and 1 lies.

4. The method according to any one of the preceding claims, characterized in that the purification of bacteriophages comprises an ion exchange chromatography and / or a benzonase treatment.

5. The method according to any one of the preceding claims, characterized in that the bacteriophage mixture contains the bacteriophage MP13.3 with the accession number DSM 18722 and the bacteriophage KP3 with the accession number DSM 18723.

6. phage mixture, characterized in that the phages have a specific activity of at least 10 ⁸ plaque-forming units per μg protein.

7. phage mixture according to claim 6, characterized in that the mixture contains the bacteriophage MP 13.3 (DSM 18722) and / or the bacteriophage KP3 (DSM 18723) with a specific activity of at least 10 ⁸ PFU / μg protein.

8. phage mixture according to any one of claims 6 or 7, obtainable by a process according to any one of claims 1 to 6.

9. Medicament, characterized in that it includes a phage mixture according to one of claims 6 to 8.

10. Medicament according to claim 9, characterized in that it additionally contains an antibiotic effective against gram-positive bacteria.

11. Medicament according to claim 9 or 10, characterized in that it further comprises an enzyme selected from the group consisting of lysines, proteases, peptidases, nucleases and / or Glucosaminidasen and / or a means for stabilizing the bacteriophages selected from human serum albumin and / or Mg ²⁺ ions.

12. Use of a phage mixture according to any one of claims 6 to 8 for the manufacture of a medicament for the treatment of infections caused by antibiotic-resistant staphylococci.

Use of a phage mixture according to any one of claims 6 to 8 for the manufacture of a medicament for preventive treatment to prevent infection caused by antibiotic-resistant staphylococci.

14. Use according to claim 12 or 13, characterized in that the infections are infections of the skin and / or mucous membranes.

15. Use according to claim 12 or 13, characterized in that the infections are caused by resistant to several antibiotics staphylococci.