DE1186981B - Manufacture and extraction of the antibiotic danomycin - Google Patents

Description

Manufacture and extraction of the antibiotic danomycin The present Invention relates to a new antibiotic called danomycin and processes for its manufacture. In particular, it relates to a Process for production by fermentation as well as separation and purification the substance. The invention encompasses the antibiotic in dilute solutions, as Raw concentrates, as purified solids and in pure crystalline form. Danomycin inhibits the growth of gram-positive bacteria, especially staphylococci, that are resistant to other antibiotics. Danomycin is non-toxic and possesses a therapeutic effect on mice infected with gram-positive bacteria. It is also valuable in treating those infected with gram positive bacteria People, for example with pneumonia. Danomycin belongs to the iron group containing antibiotics; it was originally called Antibiotic No. 425.

The present invention relates to the antibiotic danomycin, which inhibits the growth of gram-positive bacteria, is soluble in water and insoluble in acetone, has an orange-red color, shows a negative ninhydrin Tollens and Fehling reaction and in purified form at 135 to 138 ° C with decomposition melts; in water it shows an ultraviolet absorption spectrum with maxima at 270 m # t (E'1 '. = 48), 325 m # t (El l.'. = 12.6) and 430 m # t (E ' Cm = 15 , 6); the analysis gave C 48.82 ° / o, H 7.05 ° / 0, N 7.81 ° / 0, Fe 3.13 ° / 0 and 0 (as the difference) 33.190 / 0.

In addition, there is a method for producing the antibiotic danomycin The present invention provides a strain of Streptomyces albaduncus in an aqueous carbohydrate solution containing a nitrogenous nutrient cultured under submerged aerobic conditions until the solution is substantial Has efficacy against gram-positive bacteria, whereupon the danomycin from the solution is obtained.

The organism producing the antibiotic of the present invention became isolated from a soil sample and represents a new species of the genus Streptomyces which is referred to as Streptomyces albaduncus. A culture of the living organism with the laboratory designation No. 13246 was in the American Type Culture Collection, Washington, D. C., and assigned to this collection as ATCC 14698.

The strain No. 13246 of S. albaduncus has the following characteristics: 1. The aerial mycelium is short and wavy; the spore-bearers have loops or hooks and hardly form left-handed spirals. The observed with an electron microscope Spore surface is thin.

2. Czapek agar: the culture is pale yellow-brown and penetrates the agar a. The white aerial mycelium is poor and no soluble pigment is produced.

3. Glycerine Czapek Agar: The culture is small and pale yellow-brown; she penetrates the agar. The white aerial mycelium is scanty and forms pale yellow-brown soluble pigments.

4.Glycerin ammonium salt agar: the culture is small and yellowish white, and neither aerial mycelium nor soluble pigment is formed.

5. Glucose-Asparagine Agar: The pale yellow brown culture is glossy and penetrates the agar. The white aerial mycelium is powdery and an apricot yellow soluble pigment is formed.

6. Starch agar: The dark gray-brown culture is shiny and penetrating into the agar; there is neither aerial mycelium nor soluble pigment. the Starch is highly hydrolyzed. 7. Nutrient Agar: The pale yellow culture is small and penetrates the agar. Neither an aerial mycelium nor a soluble pigment occurs.

B. Bennett agar: The yellowish-olive-brown culture is moderate, the powdery or velvety aerial mycelium is coated in white or light brown; it becomes a yellowish olive brown Pigment formed.

9. Oatmeal Soyton Agar: The yellowish-olive-brown culture is moderate, the white aerial mycelium is powdery or velvety; it becomes a yellowish olive brown soluble pigment formed.

10. Potato plug: The pale yellow-brown culture is glossy, but small, and neither aerial mycelium nor soluble pigment is formed.

11. Gelatin sting: a white colony grows on the surface, and neither aerial mycelium nor soluble pigment is formed; the liquefaction the gelatin is moderate.

12. Tyrosine yeast gelatin sting: Pale yellow wrinkled colony, scanty Aerial mycelium and yellow soluble pigment were observed.

13. Milk: pale brown ring growth, no aerial mycelium and no pigment were observed; Milk is not digested.

14. Nitrate solution: A spherical, colorless mass grows on the surface, and neither aerial mycelium nor soluble pigment is formed. The rediction from nitrate to nitrite is negative.

15. Melanin Medium: The pale yellow growth is scant and invades the agar; aerial mycelia or soluble pigment are not formed.

16. The assimilation of carbon compounds was following the procedure checked by P r i d h a m; The following results were obtained: Good carbon utilization has been observed with arabinose, xylose, glucose, galactose, fructose, cellobiose, Lactose, maltose, raffinose, sorbito, inositol, mannitol, rhamnose, sodium citrate and sodium succinate. The utilization of sorbose, sucrose and inuline was doubtful or negative. When compared to a number of Streptomyces species, some resemble the S. albaduncus in some respects, for example S. albofiavus, S. pseudogriseolus and S. griseolus. However, these species can be from the present strain by the Morphology of the spore carriers due to the surface structure of the spores, the growth sites cultures and physiological or biochemical properties as described in more detail below.

The S. alboflavus shows straight and branched aerial mycelium and forms hardly any spirals; the color of the aerial mycelium on Czapek agar is yellowish white, while no aerial mycelium was found on glucose asparagus agar. He reduced nitrate too Nitrite and peptonization of milk are positive without coagulation. These properties distinguish him from the present tribe.

S. pseudogriseolus has numerous spirals in the spore carriers on, the color of the air mycelium is dull yellow overlaid with gray. Both the Both coagulation and peptonization are positive for milk. There were also differences found in the recovery of sodium citrate, raffinose and sorbitol. S. griseolus shows short straight spore carriers with wavy branches and has no typical, spirals similar to the present trunk; Differences are in the smooth spore surface, the gray or dark gray color of the air mycelium on Czapek and glucose-asparagine agar and especially in the formation of a brown, soluble pigment on growth determine organic media.

It is a general characteristic of Streptomycetes that their behavior on the culture medium suddenly changes or can be artificially changed. This is why mutants or variants are sometimes created out of the ground or through conservation received that differ considerably from the original trunk. These properties were also found in the strains according to the present invention.

When artificial mutations are created in the original strain to improve productivity, there are substantial changes in the properties of the cultures and in the color of the aerial mycelium, and the mutants also produce danomycin. There are numerous physical or chemical possibilities for generating artificial mutations, such as exposure to X-rays or UV rays or treatment with chemicals such as dichlorodiethyl sulfide. The following two strains are examples of mutations caused by exposure to X-rays or UV rays: Strain No. 13246-29, Strain No. 13246-28-4-27 Mutation process UV rays 1 x-rays Microscopic observation (like the original trunk) Glucose asparagine agar yellowish-brown culture, old gold-colored culture, white aerial mycelium, brown-white aerial mycelium, pale yellowish brown soluble old gold colored soluble lich pigment pigment Starch agar dark gray culture, light olive culture ,. white aerial mycelium, light gray aerial mycelium, no soluble pigment light olive colored soluble pigment Bennett agar dark, yellow-gray-brown yellow-brown culture, light- Culture, gray air- gray-brown air- mycelium, yellow-brown mycelium, yellow-brown soluble pigment soluble pigment Potato glucose agar yellow-gray-brown culture, yellow-olive-brown culture, gray aerial mycelium, white aerial mycelium, light yellow brown sol- old gold-colored sol- lich pigment lich pigment Thus, in addition to the above species, the soil variants thereof, the mutants caused by mutating agents such as X-rays, UV rays, chemicals, etc. and all strains isolated from soil samples are also included in the present invention, insofar as they meet the necessary requirements. Danomycin is an antibiotic that contains iron in molecule as described above. Therefore, the danomycin-producing strain was compared with various Streptomycete strains that also produce iron-containing antibiotics such as grisein, albomycin, EHT-22765 and ferrimycin.

The S. griseus, which produces gris, differs significantly from the present trunk, as the aerial mycelium has a characteristic water-green color, the spore carriers are straight and have tufts of hair and no typical spirals are formed.

Various Streptomyces species such as S. griseoflavus, S. galilaeus, S. lavendulae, S. pilosus, S. viridochromogenes, S. olivaceus, S. aureofaciens and S. polychromogenes form iron-containing antibiotics, and the individual species differ S. griseoflavus shows straight and monopodially branched spore carriers and does not form typical spirals. The color of the culture is reddish brown on Czapek agar and lemon yellow on glucose-asparagine agar.

S. galilaeus has monopodially branched spore carriers with irregular open spirals. The surface of the spores is smooth and the culture is on glycerol-Czapek agar carmine.

S. lavendulae has long, monopodially branched spore carriers short, compact, right-directed spirals, and the aerial mycelium is on the most media lavender.

S. pilosus has a hairy spore surface and forms a brown one soluble pigment.

S. viridochromogenes has numerous spirals and forms a brown one soluble pigment.

S. olivaceus has long spirals and forms an ash-gray to light olive gray aerial mycelium on Czapek agar.

S. polychromogenes has a light carmine to carmine red aerial mycelium on.

As described above, the danomycin producing strain became related to Compared species from the standpoint of taxonemia or antibiotics produced, and it was found that the present strain represents a new species of Streptomycetes, known as Streptomyces albaduncus nov. sp. was designated.

Streptomyces albaduncus will produce when grown under suitable conditions Danomycin.

A fermentation broth containing danomycin is prepared by inoculating a suitable medium with spores or the mycelium of the danomycin producing organism and then grown under aerobic conditions. For the production Danomycin can be grown on a solid medium for production However, for larger quantities, cultivation in a liquid medium is preferable.

The incubation temperature can be between 20 and 35 ° C, but is preferably between 25 and 30 ° C at neutral pH. In the submerged aerobic fermentation of the organism for the production of danomycin, the medium contains glycerol oil or a carbohydrate such as glycerol, glucose, maltose, sucrose, lactose, dextrin, starch, etc. in pure or raw state and as a nitrogen source an organic substance such as soybean meal, Distillers' solubles, peanut flour, yeast extract, corn steep liquor, etc. and, if desired, inorganic nitrogen sources such as nitrates or ammonium salts and mineral salts such as sodium chloride, potassium chloride and magnesium sulfate and buffering agents such as calcium carbonate or phosphates and traces of heavy metal salts; These additions are in Canadian Patent 513,324, in British Patents 730,341 and 736,325 and in the USA. Patents 2 691 619, 2658018, 2653899, 2586762, 2516080, 2483892, 2609 329 and 2 709 672 mentioned. Aerated submerged culture uses an antifoam agent such as liquid paraffin, fatty oil or silicone.

It can also be more than one type of carbon or nitrogen source or antifoams can be used in the production of danomycin. In general the incubation is continued until at least a few hundred mcg / ml danomycin have accumulated in medium.

For the isolation and purification of small amounts of the active compound the usual methods can be used from the fermentation liquor. For example, danomycin can be produced using a different absorption, Solubility and partition coefficients between the active compound and the existing impurities are isolated. That contained in the filtrate of the broth Danomycin is z. B. adsorbed by activated carbon, with water, aqueous methanol or aqueous ethanol and then washed with aqueous butanol or aqueous Acetone eluted. The eluates are combined and evaporated to dryness in vacuo, whereupon the raw powder is obtained. When using chromatography on activated charcoal and a fraction collector, the active compound can be further purified. By Addition of a large amount of a suitable organic solvent such as acetone danomycin can be precipitated and added to the aqueous concentrate of the active fractions be separated. The existing impurities are removed by adding an organic Solvent such as methanol precipitated to the aqueous concentrate and removed.

The extraction by organic solvents can also be used Purification of danomycin serve.

For example, the active compound can be suitably mixed by mixtures Solvents such as phenol-chloroform or benzyl alcohol-butanol can be extracted. The extracts can after washing with acidified water, alkaline water and pure water into the water with the addition of suitable non-polar organic Solvents such as ethers or hydrocarbons are transferred.

The countercurrent distribution method can also be used to purify the danomycin be applied; for example, phenol-chloroform-pH 6.0 buffer (1: 9: 10) or benzyl alcohol-butanol-n / 100-HC1-20% salt water (20 : 10: 3: 30) Obtain high purity danomycin.

Comparing the above-mentioned physicochemical and biological properties of danomycin with those of other known antibiotics, danomycin turns out to be a new antibiotic. Danomycin is similar to grisein, albomycin, ferrimycin, LA-5352 and other iron-containing antibiotics in its properties in terms of solubility in water and orange-red color. However, if one compares the antibacterial spectrum with that of the others, danomycin differs from grisein and albomycin in its activity against gram-negative bacteria, from ferrimycin against Bacillus sphericus and from LA-5352 against hemolytic streptococci: Since danomycin is mainly effective against gram-positive bacteria, it was compared by paper chromatography with other iron-containing antibiotics such as ferrimycin and LA-5352, which are also mainly effective against gram-positive bacteria. As shown below, danomycin is different from these antibiotics. Solution system Rr values Danomycin Ferrimycin LA-5352 3 ° / o NH, CI solution ............................. 0.95 0.60 0.00 to 0, 35 50% aqueous acetone .... ................... 0.85 0.65 0.60 Water ......................................... 1.00 0.05 0, 05 Butanol-Essi8Mure-H,% 0. (4: 1: 5) ............ 0.23 0.40 0.40 Butanol-Fsdgsäure-H, Cl (2: 1: 1) ............ 0.50 0.73 0.75 Ethanol-H1.0 (3: 1) with 2% NaCl ............. 0.65 0.45 0.45 In its infrared spectrum, danomycin shows characteristic absorption maxima at 1160 to 1165, 865 to 880 and 680 to 690 cm- ', which were not found in the spectrum of ferrimycin or LA-5352. If you compare the UV spectrum of danomycin with that of certain other antibiotics, danomycin differs as follows: Position of the maxima Grisein .................... 265 m # L 420 m # t Albomycin ............ ..... 270 m # t 440 mi. Ferrimycin ................. 228 mi., 319.425 m # L LA-5352 ................... none Danomycin ............ .... - 270 mt., 325,430 mp. The following examples serve to illustrate the invention without restricting it.

Example 1 Composition of the medium soybean meal ....................... 15 g dry horse blood ................. 5 g soluble starch ....................... 50 g potassium phosphate, dibasic. . . . ....... 1 g. Sodium chloride .......................... 0.5 g magnesium sulfate .................. .... 0.5 g calcium chloride. ... .................. 0.59 tap water ........................ 1000 ml pH. ................................. = 6.8 A nutrient medium (100m1) containing the above components is placed in a flask of 500m1 content -sterilized, inoculated with a culture of Streptomyces albaduncus and allowed to grow at 27 -f- 1 ° C for 8 days with shaking, the danomycin formation in the fermentation broth reaching 240 mcg / ml. Example 2 Composition of the medium soybean meal ....................... 25 g Soluble starch ................. ...... 65 g potassium phosphate, dibasic ........... 1 g sodium chloride ......................... 1 g magnesium sulfate ...................... 0.5 g calcium chloride .................... .... 0.5 g tap water ........................ 1000 ml pH .............. .................... = 6.8 A nutrient medium (100 ml) containing the above components is sterilized in a 500 ml flask with a culture of Streptomyces albaduncus inoculated and grown at 27 ± 1 ° C for 8 days with shaking, the danomycin formation in the fermentation broth reaching 440 mcg / ml.

Example 3 The fermentation broth was filtered, the pH adjusted to 7 to 8 and adsorbed by 0.5010 activated charcoal. The charcoal was washed with water and then with a 50% aqueous methanol solution and the danomycin was eluted with water saturated with n-butanol. The active eluate was concentrated to dryness and the crude, solid danomycin was dissolved in water and purified by activated charcoal chromatography. The column was washed with water and 50% aqueous methanol solution and then fractionally eluted with 60% aqueous acetone. The pure preparation thus obtained was again dissolved in a little water; by adding 10 parts by volume of acetone, the danomycin was precipitated and further extracted with 950 /, ethanol to remove minor impurities. The reddish brown danomycin was dissolved in water and extracted with a mixture of phenol-chloroform (1: 1). The extract was washed with a 0.5% aqueous solution of sodium bicarbonate, n / 100 hydrochloric acid and water and then added to water with the addition of ether and petroleum ether. A pure preparation of danomycin was obtained by freeze drying. Example 4 Danomycin can be purified by countercurrent distribution. Between the solvent system benzyl alcohol - n-butanol - n / 100-hydrochloric acid-20% aqueous sodium chloride solution (20: 10: 3: 30), danomycin was distributed around a no. 9 conical tube in 35 transfers. The contents of the active tubes were collected and a pure preparation of danomycin was obtained by freeze-drying. The danomycin thus obtained was determined to be pure by the good agreement of the distribution curves plotted as biological test values and the absorption at 420 m # L with the theoretical curve. Danomycin is an orange-reddish colored antibiotic that contains iron in its molecule. It is soluble in water, 50% aqueous ethanol, 50% aqueous methanol and solvent mixtures of phenol-chloroform (1: 1), moderately soluble in 95% methanol and 95% ethanol, but insoluble in acetone and other organic solvents. The ninhydrin, Tollen and Fehling reactions are all negative, but some ninhydrin-positive substances could be determined by paper chronomatography during the acid hydrolysis of danomycin. The countercurrent distribution using benzyl alcohol-n-butanoln / 100 HCl-20% aqueous NaCl solution (20:10 3:30) was used to obtain the pure preparation. The danomycin obtained in this way melts at 135 to 138 ° C. with decomposition.

Analysis: Found: C 48.82, H 7.05, N 7.81, Fe 3.13. Based on these analytical data and the assumption that only one iron atom is present, were the empirical formula and the molecular weight calculated with C7aHias0avNioFe and 1791.

The ultraviolet absorption spectrum of danomycin in water shows absorption maxima at 270 m # L (Eil = 48), 325 m; .L (El l '= 12.6) and 430 mu. (E, 11 = 15.6). The infrared absorption spectrum of danomycin in potassium bromide has characteristic absorption bands at the following wavenumbers on 3200 to 3280, 2920, 1720 to 1725, 1630 to 1640, 1570 to 1580, 1490 to 1500, 1455 to 1470, 1220 to 1230, 1160 to 1165, 1010 to 1040, 865 to 880, 810, 755 and 680 to 690 cm- '. When the paper strip chromatography was carried out in various solvent systems, the following RT values were obtained: Wet n-butanol .......................... 0.05 3 ° / oige aqueous ammonium chloride ....... 0.95 80% / oige phenol ........................... 0.95 50 ° / oiges aqueous acetone ................. 0.85 n-butanol-methanol-water (4: 1: 2) -f- 1.501, methyl orange ...... ............. 0.50 n-butanol-methanol-water (4: 1: 2) .... 0.30 benzene-methanol (4: 1) ..... ............. 0.05 water ................................. .. 1.0 n-butanol-acetic acid-water (4: 1: 5) ... 0.23 n-butanol-acetic acid-water (4: 1: 2) ... 0.35 n-butanol-acetic acid -Water (2: 1: 1) ... 0; 50 ethanol-water (3 : 1) + 2 ° / a sodium chloride 0.65 n-butanol-ethanol-acetic acid-water (25: 25: 3: 47) ......................... 0.85 danomycin exhibits the above properties; and in comparing these properties with those of other ferrous antibiotics such as grisein, albomycin, LA-5352, and ferrimycin, it can be distinguished from these.

The biological properties of danomycin are as follows: 1. Antimicrobial spectrum The minimum inhibitory doses of danomycin against gram-positive, gram-negative, acid-fast bacteria and fungi were determined by a serial dilution technique with agar. The results are shown in Table 1. Danomycin is effective against coagulase-positive staphylococci, including strains resistant to most antibiotics. Table 1 Minimal Test organism inhibitory concentration (mcg / ml) Escherichia coli NIHJ ............. > 50 Klebsiella pneumonia Julianelle, type A> 50 Salmonella typhi .................. > 50 Shigella dysenteriae A .............> 50 Neisseria sp. (CP-R) ............... > 50 Staphylococcus aureus FDA 209-P ..................... 0.039 Staphylococcus aureus FDA 209-P (ST-R) .............. 0.039 Staphylococcus aureus FDA 209-P (NB-R) ............. 0.078 Staphylococcus aureus No. 52-34 (TC, EM, CM, Pc, SM-R) 0.078 Staphylococcus aureus Smith strain .................... 0.078 Staphylococcus aureus 193 (Pc, SM-R) ..................... 0.078 Staphylococcus aureus 193 (Pc, SM, EM-R) ................ 0.078 Staphylococcus albus PCI 1200 A ... > 50 Sarcina lutea PCI 1001 ............ 0.313 Micrococcus flavus ................ 0.019 Bacillus subtilis PCI 219 ........... 0.039 Bacillus sphericus 122 ............. 0.02 Bacillus anthrasis 115 .............. 0.02 Bacillus cereus ATCC 10702 ........ > 50 Corynebacterium xerosis 53-K-1 .... 0.01 Streptococcus faecalis B-40203 ...... > 50 Streptococcus hemolyticus Dick strain ..................... > 50 Diplococcus pneumonia DP-3-5A ... 0.0024 Diplococcus pneumoniae Type II ... 0.0024 Mycobacterium tuberculosies v. hominis H "Rv ...............> 50 Mycobacterium tuberculosis 607 .... > 50 Aspergillus niger ..................> 50 Candida albicans ..................> 50 Abbreviations: TC = tetracycline -R = resistant EM = erythromycin CP = chloramphenicol CM = carbomycin ST = streptothricin Pc = penicillin NB = novobiocin SM = streptomycin 2: Effect of pH on antibacterial activity The effect was examined by a dilution method in liquid media in which a culture of the test organism was inoculated after one night at a 10-fold dilution. As can be seen in Table 2, the activity of danomycin is not influenced by a medium pH. Table 2 Minimal Test organism inhibitory concentration (mcg / ml) PH = 6.2IPH = 7.2IpH = 8.2 Staphylococcus sureus 209-P ................. 0.039 0.039 0.078 Staphylococcus aureus 193 0.078 0.078 0.078 Staphylococcus sureus Smith strain ........... 0.078 0.078 0.078 Bacillus subtilis .......... 0.039 0.039 0.078 3. Effect of serum on antibacterial activity. The effect was determined by diluting the broth. As can be seen in Table 3, the serum has no effect on the activity of the danomycin. Table 3 Minimal Inhibitory concentration Test organism (mcg / ml) Control with 50% serum Staphylococcus aureus 209-P 0.078 0.078 Staphylococcus aureus 193 0.078 0.078 Staphylococcus aureus Smith strain ............ 0.039 0.078 4. Antibacterial Activity of Danomycin Against Clinically Isolated, Coagulase-Positive Staphylococci The activities of danomycin and seven commonly used antibiotics were tested against sixty strains of coagulase-positive staphylococci isolated from patients at various clinics. The results are summarized in Table 4. A fairly wide spread of antibiotics against commonly used antibiotics was observed, while the spread of danomycin resistant strains was only 1.7%. Table 4 Minimal spread of resistant staphylococci (° / o) Inhibitory Concentration Dang- I myCin DSM I KM I TC CP PC EM I NB I. 100 .............. 1.7 55.2 0 52.5 0 30 0 0 100 .............. 0 10.4 0 6.8 1.7 43.5 3.3 1.7 10 .............. 0 22.4 3.3 1.7 98.5 11.7 0 1.7 1 .............. 10 12.1 93.5 32.2 0 3.3 97 52.5 0.1 .............. 87 0 3.3 6.8 0 0 0 44 0.01 .............. 1.7 0 0 0 0 11.3 0 0 Abbreviations: DSM = Dihydrostreptomycm. TC = tetracychn. Pc = penicillin G. KM = kanamycin. CP = chloramphenicol. EM = erythromycin. NB = novobiocin. 5. Toxicity The toxicity of danomycin is extremely low, the intravenous LD50 is 3250 mg / kg in mice. There were no dead animals at an interperitoneal dose of 5000 mg / kg. The chronic toxicity was examined in rats; Even with a daily intraperitoneal injection of 100 mg / kg danomycin, no negative effects on growth or behavior were found after 90 days.

6. Chemotherapeutic effect on experimental infection from mice mice were infected intraperitoneally with Staphylococcus aureus strain Smith, the vaccination dose being 100 times the pathogen's LD ", and danomycin was administered subcutaneously after the onset of infection. The medium healing dose a single injection (CD ") was determined to be 0.04 mg / kg Sodium penicillin G tested, the CD ") value of which was 0.3 mg / kg.

The antibiotic of the present invention is a valuable agent to prove the harmfulness of gram-negative bacteria, fungi, yeast and the like in the production of the enzyme amylase by fermentation of B. subtilis. So made possible the addition of from 1 to 1000 mcg / ml and preferably 10 mcg / ml of the antibiotic on the inoculated medium, the growth of the pests and their visual detection.

Claims (1)

Claim: Use of Streptomyces albaduncus for production of the antibiotic danomycin by conventional biological cultivation under submerged conditions and recovery from the fermentation liquor by known methods.