HU204698B - Process for producing pharmaceutical composition suitable for treating mammitis and other staphylococcus infection - Google Patents

Abstract

The invention relates to a composition for killing staphylococci. The composition consists of lysostaphin and at least one substance with synergistic enhancement on lysostaphin's bactericidal activity, for example, an antibiotic, a chelating agent or a membrane active agent. The invention further relates to the treatment method and prophylaxis of bovine mastitis utilizing lysostaphin.

Description

Scope of the description: 12 pages (including 1 page figure)

The present invention relates to a pharmaceutical composition for the treatment and prevention of Staphylococcal infection, in particular, Swine Fever Inflammatory Bovine Inflammation, which comprises the active ingredient Esostaphin.

Alizostaphin is a bacteriocin secreted exclusively by the strain originally identified as Staphylococcus simulans and subsequently called Staphylococcus staphyloly ticns by Schindler and Schuhardt. The preparation of hosostaphin with S. staphylolyticus is described in U.S. Patent No. 3,278,378, and in Proceedings of the National Academy of Sciences, 51,414-421 (1964). The only organism that produces lysostaphin is S. staphylolyticus (NRRL B-2628), a biovar of S. simulans (Sloan et al., 1982, Int J. System. Bacteriol. 32, 170-174). Since the name S. staphylolyticus is not included in the official list of bacterin names, the lysostaphin-producing organism was deposited as S. simulans.

Bacteriocin is a bacterial secreted protein that kills and in some cases destroys related bacteria, such as Esostaphin destroys and kills almost all known species of Staphylococcus, but is inactive against bacteria of other species. Lysostaphin isolated from a culture of conventionally cultured S. simulans (NRRL B-2628) is an endopeptidase that cleaves cross-linking of peptidoghcan poEglicin on the cell wall of Staphylococci.

Preliminary studies have shown that Ezostafin can be produced by a fermentation technique based on liquid culture of S. simulans. Such a fermentation technique is described, for example, in U.S. Patent No. 3,278,378 and in Proceedings of the National Academy of Sciences, 51, 414-421 (1964). The fermentation technique used to produce esostaphin is improved by U.S. Patent Nos. 3,398,056 and 3,594,484. These solutions improve the culture medium and inoculation technique, thereby accelerating and enhancing the fermentation of lysostaphin. S. simulans produced esostaphin as an inactive precursor with exponential growth. Aproenzimetaz is converted to an active enzyme by a prophase produced by a stationary phase culture of S. simulans.

In addition to the method described, lysostaphin can also be produced by recombinant microorganisms such as E. coli, BacElus subtiEs and B. sphaericus, which express the alisostaphin gene. In contrast to the natural production process, recombinant lysostaphin-producing strains increase Lysostaphin is produced as a fully mature active enzyme in Staphylococcus immunogenic impurities.

Bovine rhinitis is the biggest problem in the dairy industry, which is the only one in the United States (

It causes a loss of over $ 2 trillion a year in the States

In the United States, the disease is somewhat advanced in 50% of cows, which results in milk being unusable, reduced milk production, and in the event of multiple infections, the AEA is killed.

The mammary gland infection is due to infections of the mammary gland caused by Staphylococcus aureus or Streptococcus agalactiae and to a lesser extent by E. coE or other 10 Gram-negative bacteria or a combination thereof. Most Streptococcus infections can be effectively treated with standard anybiotics. However, staphylococcal mammary grafting is difficult to cure.

The usual method of preventing bovine mammary gland disease is to wash the lice daily with a disinfectant solution (J. S. McDonald, 6, 1984), which may in some cases also contain an antibiotic. Continuous antibiotic therapy should be used with caution as it may lead to the development of antibiotic-resistant strains. In the event of an infection, antibiotic infusions are administered via the breast. This type of antibiotic therapy is able to reduce the infection to such an extent that the milk produced can be sold, but usually does not lead to complete destruction of the pathogen.

In the past, antibiotic therapy had only a minor effect on staphylococcal mammary inflammation, and the infection frequently returned and became chronic. PMN) remains viable. It is believed that Staphylo5 coccus within the PMN is protected from the effects of the antibiotic and that, in the event of leukocyte death, the phagocytic Staphylococcus may be the starting point for a new culture of Staphylococcus that causes mammary inflammation

Experiments on the 3 mechanisms of phagocytosed Staphylococcus antibiotics have led to the finding that the phagocytosed Staphylococcus eEen has a beneficial effect on the use of Esostaphin (Craven et al., 29, Research in Veterinary Science 57, 1980, Craven et al. microbial Agents and Chemotherapy 618 (1982), Craven et al., 5th Comp. Immun. Microbial Infect. Dis. 447 (1982), Craven et al., 51. Jour of Dairy Research 513 (1984)). In the experiments, Ezostaphin was pre-treated in vitro to kill extraI cellular Staphylococcus before the phagocytosed Staphylococcus was treated with cloxacilEn, gentamycin or Esostaphin. The results of Craven et al. Show that Ezostaphin has no effect on mammary inflammation, since intracellular ¹ Staphylococcus remains viable 20 hours after incubation with the Ezostaphin-containing solution (51. Journal of Dairy Research 515-516 [Table 2]).

It has also been described that Ezostafin penetrates human monocytes. However, since monocytes are a cell type other than PMN, this human model is not al2

Applicable to the treatment of bovine mammary inflammation (van den Broek et al., 21, Scand. J. Immunol. 189 (1985)).

Lysostaphin has also been shown to be useful in the treatment of staphylococcal kidney abscess in mice, particularly when administered in combination with methicillin (Dixon et al., 41, Yale J. Biol. Med. 62, 1968).

In humans, lysostaphin is used to treat chronic staphylococcal infection of the nose (Quicek, Jr. et al., 22, Applied Microbiology 446 (1971)). In resistant staphylococcal infections, lysostaphin is administered systemically (Stark et al., 291. Medical Intelligence 239 (1974)). However, the practical use of systemically administered lysostaphin in both human and veterinary medicine remains uncertain and uncertain. Lysostaphin is too immunogenic to be generally used, except for topical treatment.

It has been found that lysostaphin can be surprisingly effective in the treatment and / or prevention of staphylococcal mammary inflammation, even in the chronic form of inflammation, without inducing immunogenic side effects. For prevention, lysostaphin is added to a daily bath.

It is known that lysostaphin is effective on its own, but according to the invention other bactericidal agents such as penicillin or lysozyme are added to the chickpea in addition to lysostaphin. The composition may further comprise a chelating agent, such as ethylenediaminetetraacetate (EDTA), and a mild surfactant, which may potentially kill the bacterium. Suitable mild surfactants are, for example, polyoxyethylene sorbitan ester and fatty acid ester (Tween series), octylphenoxypolyethoxyethanol (Triton-X series), n-octyl PD-glucopyranoside, n-octyl PD thio-glucopyranoside, n-decyl-3-D-glucopyranoside, n-dodecyl-β-D-glucopyranoside and biological surfactants such as fatty acids, glycerides, monoglycerides, deoxylates and deoxycholate esters.

In addition to the prophylactic effect of a broad-spectrum bath, certain components of the bath can be infused into the infected udder to infect bacteria and cure mammary inflammation. This can be achieved by using lysostaphin alone or in combination with a mild surfactant which surprisingly enhances the effect of lysostaphin against Staphylococcus by a factor of more than a thousand times. Thus, the formulation used for infusion treatment may contain penicillin and / or mild surfactant as active ingredient, with or without chelating agent.

Thus, an infusion of a therapeutically effective amount of lysostaphin, optionally administered as an additional active ingredient by the addition of a surfactant, EDTA, penicillin or other potentiating agent, may be used to eliminate staphylococcal infection. The infusion usually contains 2-400 mg of lysostaphin when no potentiating agent is used. In combination with a potentiating agent, the synergistic effect may reduce the dose of lysostaphin to about one thousandth.

The synergistic bactericidal activity of lysostaphin and penicillin can also be demonstrated in penicillinase-positive S. aereus and methicillin-resistant S. aureus (MRSA). MRSA is generally resistant to a range of antibiotics and therefore causes significant problems, especially in humans, and is difficult to eliminate. The lysostaphin / penicillin combination can also be used for severe MRSA infections that cannot be treated with standard antibiotic therapy (such as penicillin). Thus, penicillin and other active ingredients with lysostaphin may be used to treat staphylococcal infection.

Although only the anti-inflammatory activity of the lysostaphin-containing composition is shown within the scope of the present invention, it can be used against a variety of other Staphylococcus infections based on its proven activity. Thus, the composition of the present invention can be used to incorporate wound dressings or surgical implants, or in combination with other drugs or disinfectants, against staphylococcal infection. The composition can also be used to clean medical instruments and floors, beds and the like to provide a disinfected environment. They can also be used as nasal infusions to reduce intra-nasal Staphylococcus infections, as well as in the food industry to treat or pack meat, eggs, cheese and fish.

Lysostaphin may be obtained from natural sources or by recombinant techniques. Lysostaphin is preferably prepared from Bacillus sphaericus 00 containing a recombinant plasmid that synthesizes lysostaphin, since it produces a large amount of lysostaphin free of staphylococcal immunogenic material, which can be easily purified directly by accumulation in the culture medium. Various strains can be used to produce lysostaphin, but Bacillus sphaericus transformant containing plasmid pBCl6-II is most preferred. Techniques for producing lysostaphin from a microorganism transformed with a recombinant plasmid containing the gene encoding lysostaphin are known.

Figure 1 is a chromatogram of lysostaphin produced by B. sphaericus 00 containing the recombinant plasmid pBC16-II, which encodes lysostaphin.

For the prevention of bovine mammary inflammation, lysostaphin can be used in the bath phoma. A lysostaphin-containing bath can be used effectively to prevent bovine mammary inflammation when applied before and after each milking. Preferably, the preventive treatment is performed on all cows in the tester. The bath contains about 1.0 micrograms / ml lysostaphin with a suitable vehicle. In addition to lysostaphin, the bath of the present invention may contain as active ingredient about 0.1 micrograms / ml penicillin, about 10 micrograms / ml lysozyme and a mild surfactant.

The carrier is preferably a pH 8.0 buffer, such as an aqueous buffer or a hydrophilic ointment base. Other carriers include nonionic deteigens, fatty acids, or other mild surfactants, proteins such as serum albumin or gelatin, dotted cellulose. The infant formula produced according to the invention preferably further comprises a chelating agent such as EDTA, a coloring agent and a wetting agent such as glycerol or sorbitol.

Preferably, the lysozyme is prepared from a hen's egg protein.

Despite preventive treatment, animals with chronic or acute staphylococcal bovine mammary inflammation can be effectively treated by infusion of lysostaphin into the breast. With a dosage unit containing 2-400 mg of lysostaphin per mammary gland, infection can be eliminated and most cases of cure for staphylococcal breast inflammation are possible. In case of continuous infection, a higher dose of lysostaphin is used. However, administration of doses substantially in excess of 400 mg may result in undesirable and potentially harmful side effects such as transient swelling, tenderness and reduced milk production. However, these effects are limited to the treated gland and can be treated with a higher dose of a given gland in severe or life-threatening cases. In life-threatening cases, the treatment may be based on the ischemic effect, independently of the infected gland, for example by intravenous, subcutaneous, intramuscular, intrathecal or oral administration, in which the lysostaphin is protected from intestinal inactivation.

It has also been found that the infusion of a combination of lysostaphin and penicillin achieves a significantly greater effect than that of hosostaphin alone, since the combination produces a synergistic bactericidal effect. Thus, the lysostaphin-containing composition may further comprise agents that increase the bactericidal activity of lysostaphin, such as synthetic penicillin or other antibiotics, chelating agents and mild surfactants (e.g., 4-deoxycholate) and other membrane-active substances that accelerate the The dose of lysostaphin in a composition containing, for example, penicillin may be reduced due to the increased bactericidal activity of lysostaphin. Because needle 4 high doses of lysostaphin can cause undesirable and potentially harmful side effects, the synergistic effect is significant not only in terms of enhancing the effect but also in terms of avoiding side effects.

· 5

1-4.példák

The effect of lysostaphin and lysozyme preparation on S. aureus and other bacterial inflammatory bacteria was determined in vitro. The experiment is described below

Examination of living cells

1. Bacterial cells (typically ^{10 9} cells / ml) from overnight plate incubated at 37 ° C are suspended in Tris buffer (20 mM Tris pH 8). 6 (

2.1 µL of the bacterial cell suspension and 1 ml of the control and test infusion (i.e. milk, buffer or buffered detergent containing lysostaphin) are combined.

3. The cells are incubated at 37 ° C for various times.

4. Centrifuge the bacterial suspension in a microcentrifuge for 2 minutes to precipitate the cells.

5. Wash the resulting pellets with 0 ml phage buffer (2 x 1.0 ml).

6. The cells are resuspended in 1.0 ml of phage buffer and then diluted to the desired value in phage buffer and plated on 10 microliters of GL agar (S. aureus, E. coli, Klebsiella pneumoniae) or tryptase soy agar (S. agalactiae). 7. Incubate overnight at 37 ° C, and determine the percentage of colony forming units (CFU) and the percentage survival from the control and test plates.

) Composition of phage buffer:

Tris (pH 7.8) mmol / L magnesium sulfate

4 mmol / l calcium chloride 100 mmol / l sodium chloride 1.0 g / l gelatin

Phage buffer stabilizes non-degraded pro-plastics and spheroplasts during treatment

GL agar composition:

Per liter, 3.0 g Difco casino acid, 3.0 gf Difco yeast extract, 5.9 g sodium chloride, 3.3 ml 60% w / v sodium lactate, 4.0 ml 25% glycerol, 15 g agar, pH = 7.8.

Triptych soy composition:

Per liter, 15 g Bacto tryptone, 5 g Bacto soot, 5 g sodium chloride, 15 g agar, pH 7.3.

The results of in vitro experiments demonstrating the bactericidal activity of various formulations of lysosilaphin are shown in Tables IA-IC. The results give the percentage survival of S. aureus Newbold 305, strain RN45I, penicillin-resistant strain RN1753 (producing penicillinase) and Col (methicillin-resistant) strain.

Table IA shows the results of formulations containing 1 micrograms / ml, 0.1 micrograms / ml, 0.01 micrograms / ml and 0 micrograms / ml (control) lysostaphin. The results show that all levels of lysostaphin tested were effective in killing the organism in the buffer vehicle (50 mM Tris pH 8.0). Concentrations of only I micrograms / ml and 0.1 micrograms / ml reduce the bacterial survival in the dairy

Table IB shows the effect of a formulation containing lysostaphin and a mild nonionic surfactant, octylphenoxypolyethoxy (10) ethanol (Triton-X-100). For example, in formulations containing 0.1 micrograms / ml lysostaphin and 0.1 wt% Tnlon-X-100, less than 0.001% of the cells survive, compared to 2.2% and 7.7%. More surprisingly, survival is less than 0.001% at 0.01 micrograms / ml li4

EN 204,698 Β for zostafin and 0.1 wt% Triton-X-100.

Table IC shows the synergistic effect of the lysostaphin / penicillin combination in three Staphylococcus strains. Depending on the dose, the lysostaphin / penicillin combination is 100-1000 times more potent than lysostaphin or penicillin alone in each of the three strains.

Table ID shows the effect of the lysostaphin / penicillin combination versus the sequential action of the active compounds in S. aureus. S. aureus was suspended in milk at a concentration of 10 ⁷ cells / ml and incubated with lysostaphin followed by penicillin or a combination thereof for the period indicated in the table. After incubation, the samples are centrifuged, the cell pellet washed twice, suspended in 1.0 ml of phage buffer, diluted and transferred to 100 microliters of GL agar plate.

Incubate overnight at 37 ° C and determine the percentage survival relative to the control based on the number of colony forming units (CFU). The lysostaphin / penicillin combination exhibits at least three orders of magnitude na10 greater synergistic bactericidal activity against S. aureus compared to the sequential treatment of the two active ingredients.

IA footer

Effect of lysostaphin on S. aureus

Tribe Carrier Incubation time Survival (%) control 1, OL 0, lL 0.0 IL S. aureus Newbould 305 milk 15 minutes 2.8 75.0 100 100 2 o'clock 0.1 82.0 100 100 RN451 milk 15 minutes <0.1 22 100 100 2 o'clock <0.01 41 100 100 buffer 2 o'clock - 2.2 20 IOO

Table IB

Non-ionic detergent enhanced lysostaphin bactericidal activity against S. aureus

Tribe Carrier incubation time Survival (%) control 0.1L 0.01L 0.1% T O, 1L + 0.1% T 0,0IL + 0.1% T S. aureus buffer + 0.1% RN451 Triton 2 o'clock 2.2 20 7.7 0,001 0,001 100

Table IC

Penicillin enhanced bactericidal activity of lysostaphin on S. aureus

Tribe Carrier Incubation time Survival(%) control O, 1L 0,01L O, 1P O, IL + 0.1P 0.01 L + 0.1P S. aureus milk 30 minutes 19 100 76 2.8 45 100 RN451 2 o'clock 26 100 17 <0.01 0.4 100 RN1753 is a penicillinase positive milk 2 o'clock 1.9 66 (10P) 46 (10P) <0.01,100 (10P) 14 100 Col methicillin resistant milk 2 o'clock 1.0 100 (10P) 67 (10P) <0.01 (10P) 0.5

Table 1D

Comparison of the Effect of the Lysostaphin / Penicillin Combination on the Effects of Successive Drugs on Staphylococcus aureus RN451 in Milk at 37 ° C

Survival (%) Combination (2 hours) 0.0005 Ispn (2 hours) 23 pen (2 hours) 25 pen (2 hours) Ispn (0.5 hours) . 0.3 Ispn (2 hours) pen (0.5 hours) 10

Comment:

Ispn = lysostaphin pen = penicillin

By further examining the activity of A. lysostaphin and lysozyme, a cloud of suspensions of live cells of S. aureus, S. agalactiae, E. coli and Klebsiella pneumoniae was measured at 600 nm. A decrease in turbidity indicates that a chelating agent (such as EDTA) enhances the activity of the three bacteriolytic enzymes

The results of the measurements indicate that lysostaphin is a fast-acting and highly effective staphylocide which can be increased several times more by the action of penicillin or mild surfactant. The bactericidal effect of lysostaphin can be further enhanced by the addition of a chelating agent. The results further show that the activity of lysostaphin can be enhanced by synthetic penicillins and cell wall-active antibiotics. Alisostaphin has the effect of staphylocid in milk, but the potency of bactericide should be approximately tenfold.

Example 5

1-4. Further general in vitro experiments were carried out to investigate the bactericidal activity of lysostaphin preparations containing the bacteriolytic enzyme, the nonionic detergent and the buffered chelating agent. As shown in Table H, 1% by weight Triton-X-100, 0.1 micrograms / ml lysostaphin, 10 micrograms / ml lysozyme and 5 mM EDTA in 20 mM Tris pH = 8.0) (AMBI Tea Dip-0.1) .Especially effective against a variety of mammary germs such as S. aureus Newbold 305, S. epidermidis, Streptococcus agalactiae McDonald and C48, and cytinically isolated Streptococcus uberis, E. coli and Klebsiella pneu35 strains.

II. spreadsheet

In vitro activity of AMBI TeatDip-1 against pathogens of breast inflammation

Tribe É15 cell count Survival (%) Staphylococcus aureus (Newbold 305) 7,0x10 * <0.001 Staphylococcus aureus (RN45I) 5,7x10 * <0.001 Staphylococcus epidermidis (PS) 8,3X10 * <0.001 Streptococcus agalactiae (McDonald) 3,9x10 * <0.001 Streptococcus agalactie (C48) 2.9x10 ⁴ <0.001 Streptococcus uberis (PS) 6,9x10 * <0.001 Escherichiacoli (PS) 9, lxl0 * <1.0 Klebsiella pneumoniae (PS) 9,6x10 * <1.0

HU 204 698 B

Example 6

The in vivo effect of the lysostaphin-containing bath in cows is investigated. The test is generally performed according to National Mastitis Council A prescription. Accordingly, the baits are cleaned with 1% iodine wash solution and dried on a paper towel. Afterwards, rinses are rinsed with alcohol and allowed to air dry. The four goats were submerged in a suspension of 10 ⁸ cells / ml of S. aureus Newbold 305 strain and air-dried for 30 minutes. Two baits (right front and left rear) are immersed in the test lysostaphin bath (10 micrograms / ml lysostaphin in 0.85% w / w saline) so that the baffle is partially covered by two thirds. It is then air dried for 30 minutes. Untreated two goats were used as controls. The litters are first wiped with a damp wool cloth, then washed with 10 ml of 0.85% sterile saline, and the wash is collected in a sterile 30 ml tube. A 0.2 ml sample of the wash liquid and its corresponding dilution were transferred to a blood agar plate in duplicate and incubated for 24-48 hours at 37 ° C. The percentage survival of S. aureus relative to the control is determined by counting the number of colonies.

The surface of the beak may be completely disinfected with S. microfluidine solution from S. aureus at 10 micrograms / ml. When lysostaphin is applied to the surface of the beak prior to immersion in S. aureus suspension, it has a satisfactory residual effect and prevents colonization. The residual effect may be enhanced by the use of polymeric adsorbent and / or inhaled carrier protein as these reduce lysostaphin leaching.

Example 7

Based on the results of Example 6 and the data obtained in the tendon staining experiment, an improved bath composition (AMBI Tea Dip 1.0) was prepared against S. aureus Newbold 305 strain containing 1.0 micrograms / ml lysostaphin, 10 micrograms / ml lysozyme, Containing 0 wt% Triton-X-100 and 5 mM EDTA in 20 mM Tris pH 8.0. The infants were immersed in a suspension of 10 ⁸ cells / ml S. aureus Newbold 305 and air-dried for 30 minutes. The treated chicks are then immersed in AMBI Tea Dip 1.0 and air-dried for 30 minutes. It is then wiped with a damp wool cloth and rinsed with 10 ml of sterile 0.85% saline. A sample of the cloth and rinse fluid was transferred to a blood agar plate separately and incubated for 2448 hours. The CFU number is determined. The results, shown in Table IIIA, clearly show the efficacy of the preparation. The number of S. aureus in treated broods is reduced by at least three orders of magnitude, and 50% of treated broods are S. aureus free.

The above assay was repeated by immersing the pups in a suspension of twice 10 ⁷ cells / ml of the strain of Streptococcus agalactiae McDonald and air-drying for 30 minutes. The measurement results are shown in Table IIIB. All treated chicks are free from S. agalactiae.

Table IIIA

Effect of AMBI Tea Dip 1.0 against Staphylococcus aureus

Cow serial number Control CFU / ml Control CFU / ml LF RH LF RH First 225 1.675 13 0 Second 24,500 19,500 8 175 Third 300 15,000 0 15 4th 78 155 0 150 5th 50.500 18,750 5 8 6th 44.250 65,500 0 0 7th 75 43 35 3 8th 175 1,150 0 0 9th 68 58 0 5 10th 550 300 0 0 Average 12.072 12.213 6 36 Sum. negative 0/10 0/10 6/10 4/10

HU 204698 B 2

Table IIIB

Effect of TeatDip 1.0 in against Straptococcus agalactie

Cow number Control CFU / ml Treated CFU / ml LF RH LF RH First . 5 15 0 · 0 Second  53 360th 0 0 Third 115 48 0 0 4th 150 10 - 0 She 5th 13,750 1,200 0 0 6th 16.250 - 725 0 . 0 7th 95 320 0 : She 8th 0 '450 0 She 9th 1.175 775 She 0 10th 150 300 0 0 Áűag 2.574 420 = 'θ ⁷ 0 Sum. negative 1/10 0/10 10/10 / ίο / w;

8.példa

The guinea-pig moth is infected with S. aureus Newbold 305 strain 200-300 CFU. Three days after infection, 200 microliters of a lysostaphin unit in 0.85% sterile saline was infused into the glands. Milk samples are taken from the amygdala 6 hours after treatment, and sampling is continued for 30 hours every 12 hours for at least 5 days. 100 microliters of milk from the treated and untreated infected glands is plated on a blood agar plate. After incubation for 24-48 hours, the plates were counted for CFU. The dose of lysostaphin required to eliminate infection did not cause any adverse side effects, suggesting that hosostaffn is well suited for the treatment of mammary staphylococcal inflammation. At a dose of 125 micrograms / ml, the glands are free of infection 6 hours after treatment and throughout the study; 40 remain free.

Γ7. spreadsheet

Effect of intravenous infusion of lysostaphin on guinea pig staphylococcal mammary inflammation

Lysostaphin dose (microgram / kg) 0 1.0 5.0 25.0 62.5 125.0 are infected zéstől exempt animals S2áma (0/10) (1/0) (1/2) (2/2) (you) (7/7)

hey surfactants or chelating agents. _T

According to the invention, lysostaphin can be obtained from natural sources and recombinant techniques. In a preferred embodiment of the preparation of lysostaphin, a culture of recombinant plasmid transformed Bacillus sphaericus 00, suitable for direct lysostaphin synthesis, is started as described in U.S. Patent Application Ser. Partially Continued Notification. The process yields a large amount of lysostaphin substantially free of staphylococcal immunoglobulin contamination. Purification of lysostaphin is facilitated by the accumulation of active lysostaphin directly in the medium. Various strains of bacillus may be used as sources of lysostaphin, but Bacillus sphaericus 00 (B. sphaericus 00 / pBC161L) containing the plasmid pBC11-1L is most preferred. ;

The lysostaphin-producing organism is cultured under the conditions necessary for lysostaphin production. . The optimum conditions will vary depending on the strain, but some i · medium and fermentation conditions required for lysostaphin production are known. For the Bacillus sphaericus 00 / pB Cl 6-1L transformant, VY medium (25 g calf infusion and 5 g yeast extract / l) is preferably used with thorough aeration (see Table V).

(Table on next page)

The examples show that lysostaphin can be successfully used in the treatment of staphylococcal mammary inflammation and its effect can be greatly enhanced when alizostaphin is used with penicillin or other substances such as mild

HU 204 698 B

Table V.

Effect of aeration on the production of Bacillus sphaericus 00 / pBC16-lL transfective lysostaphin

Mixing speed Klett 100 turns 200 turns 200 turns (ripples) 320 turns 250 21.8 36.2 35.9 30.0 350 40.1 68.9 45.3 45.0 400 88.5 62.7 102.8 71.4 450 So 86.4 52.3 135.9 YOU 64.4 31.3 37.6 57.5

The culture (40 ml) was inoculated with 300 ml Klett flask with 4 ml overnight primary culture. VY medium containing 5 micrograms / ml erythromycin was used as medium.

Samples are occasionally removed during culture. In the supernatant, the activity of lysostaphin was measured by turbidometric clarification of dead S. aureus cells. Results are expressed as micrograms / ml.

Transforming B. sphaericus 00 / pBC16-lL cultivated on VY medium produces and excretes approximately 130 mg lysostaphin per liter of medium, which is more than four times the amount produced by fermentation with S. simulans under the most favorable conditions. Lysostaphin accumulates in the medium with little or no degradation over a long incubation period and accounts for more than 80% of the total extracellular protein.

Lysostaphin can be recovered from the culture medium by the conventional fractionation precipitation method. Preferably, purification is accomplished by a combination of the precipitation method and chromatographic separation from the fermentation broth of B. sphaericus 00 / pBCl6-11L transformant producing lysostaphin.

For example, cells are removed from the fermentation broth by centrifugation or ultrafiltration and 40-60%, preferably 50%, solid sodium sulfate is added to the supernatant. After 1 hour at 4 ° C, the precipitate containing lysostaphin was removed by centrifugation. During the process, the rate of excavation is over 80%.

The precipitate was dissolved in a minimal volume of 10 mM sodium phosphate buffer, pH 7.00, 50 mM sodium chloride, and dialyzed against 100 volumes of the above buffer. After removal of the particulate material, the dialyzed solution is chromatographed on a cation exchange column (preferably Pharmacia FPLC Mono S) with 0.05-0.25 M sodium chloride. eluted with increasing buffered gradient. The recovery of lysostaphin by a chromatographic step is more than 90%. Lysostaphin activity is associated with two major peaks (Figure 1). The later eluting peak contains the non-covalent aggregate of the protein. These aggregates are dissociated in buffer and dissociated under sodium docedyl sulfate polyacrylamide gel electrophoresis conditions.

Preferably, the lysostaphin-producing strain Bacillus sphaericus is produced by recombinant DNA techniques as described in U.S. Patent Nos. 852,407 and 034,464. This involves partial digestion of the complete DNA of S. simulans with appropriate restriction endonucleases and ligation of the resulting DNA fragments to the known PUC8 linearized vector carrying the antibiotic-resistant maimer and the lac Z 'gene (i.e., β-galactosidase gene). The ligation mixture was transformed into E. coli strain JM105. Insertion of the lysostaphin gene into the plasmid is verified by culturing the transformants with an appropriate antibiotic and screening for the lac-Z 'negative phenotype. Lysostaphin production is detected by turbidometric clarification of the suspension of S. aureus as a solution or as an agar plate layer.

By using various lysostaphin-producing E. coli JM105 transformants, and restriction analysis and subcloning of plasmid JM105 DNA, the DNA sequence encoding lysostaphin is located on the 1.5 Kbp Hpa II-Hind III DNA fragment. This fragment can be visualized after electrophoresis by ethidium-biOmid staining and applied to a u-cell filter strip. The strip was washed with NET buffer (0.15 M sodium chloride, 0.1 M EDTA, 0.02 M Tris, pH 8.00) and in NET buffer containing 1 M sodium chloride. After incubation at 65 ° C for 1 hour, the transferred DNA was eluted. Ethidium bromide is removed from the DNA by extraction with n-butanol. Two volumes of cold 95% ethanol were added to the aqueous phase, the DNA was precipitated, centrifuged, washed with 80% ethanol and in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). dissolved. In order to express lysostaphin, a derivative of plasmid pBCl6 (pBCl6-lL) was used as a cloning vector to produce a recombinant plasmid transformed with B. subtilis or B. sphaericus. PBC16 is a tetracycline resistant (Tet ¹ ) plasmid Bacillus isolated from B. cereus (K. Bemhard, H. Sehremph, and W. Goebel, 133 J. Bact. 897, [1978]). Plasmids that cannot be distinguished from pBC16 by restriction analysis or Southem hybridization can be isolated from B. subtilis and B. sphaericus (J. Polak and RN Novick: 7, Plasmid, 1982).

HU 204698 Β

The pBC1I1 derivative (pBC16-1) used as a blinding vector was constructed by constructing the TaqlA strand of plasmid pE1994 (B. Weisblum, Y. Graliam, T. Gryczan and D. Dubnau, 137, J. Bact 635, [1979]). Erythromocin-resistant (emi) plasmid from S. aureus is digested and plasmid pBC16 is partially digested with Taq1 using T4 Egase. The Eugene mixture was transformed by protoplast transformation (S. Chang and S.

N. Cohen: 168, Molec. Gene. Gene 111 (1979)) was introduced into B. subtiEs and 10 clones resistant to tetracycline and erythromycin were selected. One of these clones receives a signal from pBCl6-l.

By restriction analysis, pBC16-I contains all the pBC16-Taq fragments and the Pel94 Taq1A fragment carrying the erythromycin-resistant origin. PBCl6-1 is digested with restriction endonuclease Pvu H, whereby about 25% of the plasmid DNA, which contains most of the tetracycline resistant derivative, is removed. Pvu H digested pBC16-l vector was treated with alkaline calf intestinal phosphatase. The 1.5 Kbp DNA fragment encoding lysostaphin was treated with the Klenow fragment of DNA polymerase. The 1.5 Kbp DNA fragment and the plasmid DNA were mixed and digested with T4 B gas, and the digested mixture was introduced into B. subtype Esb by protoplast transformation. The transformant is erythromycin resistant, tetracycline sensitive, and produces lysostaphin, as shown by the clear bands grown on agar plates containing dead S. aureus cells. One such Esostaphin-producing clone is called B. subti-30 Es / pBCl6-II. Plasmid pBCX6-II extracted from B. subtilis / pBCl6-II transformant was isolated after ultracentrifugation with a density gradient of ethidium bromide / cesium chloride. Plasmid pBCl6-IL was introduced by protoplast transformation into various Bacillus cells such as B. sphaericus 00. The transformants are erythromycin-resistant and produce lysostaphin. The majority of Esostaphin is accumulated in B. sphaericus 00 / pBC16-lL transformant termeh and in intact, enzymatically active form. AB. 40 strains of sphaericus 00 were originally isolated from soil and a

Reserved at the Public Health Research Institute, New York (RN3106). B, sphaericus 00 / pBCl61L is maintained by PubEc Health Research Institute of New York and deposited with the American Type Culture Collection under ATCC No. 67,398.

Claims (7)

PATENT CLAIMS CLAIMS 1. A process for the manufacture of a medicament for the treatment of bovine mastitis and other Staphylococcus infections, characterized in that 0.01 to 2.0 pg / ml of Ezostafin is produced in a manner known per se and 0.05-0.2 pg / penicillin, 520 pg / ml lysozyme, 0-10 mmol / l chelating agent such as EDTA and / or 0-2.0% mild surfactant such as octylphenoxy-phthoxyethanol ethanol with a pharmaceutically acceptable carrier and and / or other pharmaceutical excipients, and the mixture is formulated for administration. 2. The method of claim 1, wherein the concentration of lysostaphin is at least 0.01 pg / ml. 3. The method of claim 1, wherein the penicillin is 0.1 µg / ml. 4. A process according to claim 1 wherein 0.1 to 1.0% by weight of mild surfactant is used. 5. The method of claim 1, wherein 0.1 pg / ml penicillin and 0.1-1.0% mild surfactant are used. 6. The method of claim 1, wherein the eposafin is produced from a transformed microorganism comprising a recombinant plasmid encoding lysostaphin. 7. The method of claim 6, wherein the eosostaphin is prepared from a transformed microorganism containing the plasmid pBC16-IL.