U.S. patent application number 09/968598 was filed with the patent office on 2002-08-29 for tyrissamycin antibiotic.
Invention is credited to Cutrone, Jingfang, Foster, Lyndon M., Krampitz, Kimberly, Mamber, Stephen W., McClure, Grace, Peterson, Todd C., Rupar, Lisa C., Thompson, Katie A..
Application Number | 20020119145 09/968598 |
Document ID | / |
Family ID | 26792930 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020119145 |
Kind Code |
A1 |
Cutrone, Jingfang ; et
al. |
August 29, 2002 |
Tyrissamycin antibiotic
Abstract
A novel tyrosyl diester antibiotic is obtained from fermentation
of a recombinant strain of Streptomyces lividans designated
Stretomyces lividans WD 15684 (ATCC-202143). The new antibiotic,
designated tyrissamycin, exhibits antibacterial activity,
particularly against gram-positive bacteria.
Inventors: |
Cutrone, Jingfang;
(Wallingford, CT) ; Foster, Lyndon M.; (Carlsbad,
CA) ; Krampitz, Kimberly; (Southington, CT) ;
Mamber, Stephen W.; (Wethersfield, CT) ; McClure,
Grace; (Northford, CT) ; Peterson, Todd C.;
(Coronado, CA) ; Rupar, Lisa C.; (Woodbury,
CT) ; Thompson, Katie A.; (Milford, IA) |
Correspondence
Address: |
Bristol-Myers Squibb Co.
5 Research Parkway
Wallingford
CT
06492
US
|
Family ID: |
26792930 |
Appl. No.: |
09/968598 |
Filed: |
October 1, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09968598 |
Oct 1, 2001 |
|
|
|
09378309 |
Aug 20, 1999 |
|
|
|
60097214 |
Aug 20, 1998 |
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Current U.S.
Class: |
424/115 ;
435/253.3 |
Current CPC
Class: |
C07B 2200/13 20130101;
C12N 1/205 20210501; C12P 1/04 20130101; C12P 7/62 20130101; C07C
69/28 20130101; C12R 2001/465 20210501 |
Class at
Publication: |
424/115 ;
435/253.3 |
International
Class: |
A61K 035/00; C12N
001/20 |
Claims
What is claimed is:
1. A process for the preparation of tyrissamycin having the
following characteristics: (a) a white amorphous powder; (b)
soluble in chloroform, dimethylsulfoxide and methanol, and
essentially insoluble in water: (c) exhibits an ultraviolet
absorption spectrum when dissolved in methanol substantially as
shown in FIG. 1; (d) exhibits an infrared absorption spectrum (KBr)
substantially as shown in FIG. 2; (e) exhibits a proton magnetic
resonance spectrum in CH.sub.3OD substantially as shown in FIG. 3;
and (f) exhibits a .sup.13C magnetic resonance spectrum in
CH.sub.3OD substantially as shown in FIG. 4 which comprises
cultivating a tyrissamycin-producing strain of Streptomyces
lividans under submerged aerobic conditions in an aqueous nutrient
medium containing assimilable sources of carbon and nitrogen until
a substantial amount of tyrissamycin is produced by said organism
in said culture medium.
2. The process of claim 1 which includes the step of isolating
tyrissamycin from the medium in substantially purified form.
3. A biologically pure culture of Streptomyces lividans
ATCC-202143, said culture being capable of producing the compound
tyrissamycin as defined in claim 1 in a recoverable quantity upon
submerged aerobic cultivation in an aqueous nutrient medium
containing assimilable sources of carbon and nitrogen.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This divisional application claims the benefit U.S.
Non-Provisional Application Serial No. 09/378,309 filed Aug. 20,
1999 and U.S. Provisional Application Serial No. 60/097,214 filed
Aug. 20, 1998.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to a novel tyrosyl diester
antibacterial antibiotic designated by the present inventors as
tyrissamycin which may be obtained by cultivation of a strain of
Streptomyces lividans. The antibiotic provided by the present
invention has antibacterial activity against a variety of
gram-positive bacteria.
[0004] 2. Background Art
[0005] The present inventors are not aware of any tyrosyl diester
compound in the literature having a similar structure to
tyrissamycin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 shows the ultraviolet absorption spectrum of
tyrissamycin in methanol.
[0007] FIG. 2 shows the infrared absorption spectrum of
tyrissamycin (KBr pellet).
[0008] FIG. 3 shows the .sup.1H NMR spectrum of tyrissamycin in
CH.sub.3OD.
[0009] FIG. 4 shows the .sup.13C NMR spectrum of tyrissamycin in
CH.sub.3OD.
SUMMARY OF THE INVENTION
[0010] The present invention provides the novel antibacterial
antibiotic designated by the present inventors as tyrissamycin and
a fermentation process for production of this antibiotic using a
novel recombinant strain of Streptomyces lividans designated herein
as Streptomyces lividans WD 15684 (ATCC-202143). The antibiotic of
the present invention has been found to be useful for the treatment
of a variety of bacterial diseases caused by gram-positive
bacteria.
[0011] Also provided are pharmaceutical compositions of
tyrissamycin, a method for the treatment of bacterial diseases in
animals using tyrissamycin or a pharmaceutical composition thereof,
and a fermentation process for obtaining the antibiotic.
DETAILED DESCRIPTION OF THE INVENTION
[0012] The tyrissamycin antibiotic of the present invention may be
prepared by fermentation of a tyrissamycin-producing strain of
Streptomyces lividans. The preferred producing organism is a novel
strain of Streptomyces lividans TK-64 designated herein as
Streptomyces lividans WD 15684. The organism was isolated as a
recombinant streptomycete using the procedures described in
published application WO 96/34112 and in Chapter 12, pages 71-76,
Proceedings from the Genetics and Molecular Biology of Industrial
Microorganisms (GMBIM) Conference, 1997, edited by Richard H.
Baltz, George D. Hegeman and Paul L. Skatrud. Briefly, DNA was
isolated from one or more actinomycetes, none of which were
Streptomyces lividans. The actinomycete DNA was inserted into
plasmids which were used to transform Streptomyces lividans TK-64.
A biologically pure culture of the resulting recombinant strain WD
15684 has been deposited with the American Type Culture Collection,
10801 University Boulevard, Manassas, Va. 20110-2209, and added to
its permanent collection of microorganisms as ATCC-202143. The
culture is also maintained as a dormant culture in lyophile tubes
and cryogenic vials in the Bristol-Myers Squibb Pharmaceutical
Research Institute Culture Collection, 5 Research Parkway,
Wallingford, Conn. 06492. The results of taxonomic studies
performed on strain WD 15684 confirm that it is a strain of
Streptomyces lividans.
[0013] It is to be understood that for the production of
tyrissamycin the present invention, although described in detail
with reference to the preferred producing strain Streptomyces
lividans WD 15684, is not intended to be limited to this
microorganism. It is specifically intended that the invention
embrace strain WD 15684 and all tyrissamycin-producing variants and
mutants thereof which may be obtained by methods well known to
those skilled in the art, eg. by subjecting the deposited
microorganism to x-rays or ultraviolet radiation, nitrogen mustard,
phage exposure, and the like.
[0014] The tyrissamycin antibiotic may be produced by cultivating a
tyrissamycin-producing strain of Streptomyces lividans, preferably
a strain having the identifying characteristics of Streptomyces
lividans ATCC-202143, or a mutant or variant thereof, under
submerged aerobic conditions in an aqueous nutrient medium
containing assimilable sources of carbon and nitrogen until a
substantial amount of tyrissamycin is produced by said organism in
said culture medium. The tyrissamycin so obtained may be isolated
from the fermentation medium in substantially pure form by
conventional procedures such as those described in the examples
which follow. Tyrissamycin exhibits antibacterial activity in
standard antibacterial model systems.
[0015] The producing organism is grown in a nutrient medium
containing an assimilable source of carbon such as glucose,
cellobiose, trehalose, potato starch, glycerol or ribose. The
medium should also contain an assimilable source of nitrogen such
as fishmeal, peptone, peanut meal, cottonseed meal or cornsteep
liquor. Nutrient inorganic salts can also be incorporated in the
medium so as to provide sodium, potassium, ammonium, calcium,
phosphate, nitrate, chloride, bromide, carbonate and like ions.
Trace elements such as copper, manganese, iron, zinc, etc. are
added to the medium if desired, or they may be present as
impurities of other constituents of the medium.
[0016] Production of the tyrissamycin antibiotic may be effected at
any temperature conducive to satisfactory growth of the organism,
i.e. 22.degree. C. to 42.degree. C., and is conveniently carried
out at a temperature of about 32.degree. C. The fermentation may be
carried out in shake flasks or in laboratory or industrial
fermentors of various capacities. When tank fermentation is to be
used, it is desirable to produce a vegetative inoculum in a
nutrient broth by inoculating a small volume of the culture medium
with a slant, a cryopreservative culture or a lyophilized culture
of the producing organism. After obtaining a viable and active
inoculum in this manner, it is transferred aseptically to the
fermentation tank charged with production medium for large scale
production of tyrissamycin. The medium in which the vegetative
inoculum is prepared can be the same as or different from that used
in the tank as long as it is such that a good growth of the
producing organism can be obtained. Further agitation may be
provided by a mechanical impeller. Antifoam agents such as lard oil
or silicone oil may also be added if needed. Antibiotic production
may be monitored by thin layer chromatography (TLC) assay, TLC
bioautographic assay, column chromatographic assay, high
performance liquid chromatography (HPLC) assay or by conventional
biological assay.
[0017] When the fermentation is complete, the tyrissamycin
antibiotic is extracted from the culture broth with a suitable
organic solvent such as ethyl acetate or 1-butanol and the
antibiotic recovered by the extract and purified by conventional
isolation procedures.
[0018] Physico-chemical Properties
[0019] A purified sample of tyrissamycin was isolated as a white
amorphous powder. Other characterizing properties of the antibiotic
are as follows:
1 Solubility: Soluble in chloroform, dimethylsulfoxide (DMSO), and
methanol and practically insoluble in water. Ultraviolet Spectrum:
UV max MeOH (Absorbtivity): 212 (4.7), 219 (sh. 4.6), 278 (1.6) nm
(FIG. 1) Infrared Spectrum: Major Bands (cm.sup.-1) 2925, 2854,
1742, 1711, 1517, 1465, 1237 cm.sup.-1 (FIG. 2) .sup.1H-NMR: See.
FIG.3 (CH.sub.3OD) .sup.13C-NMR: See FIG.4 (CH.sub.3OD)
[0020] Based on the above properties, the structure of tyrissamycin
is believed to be 1
[0021] n.sub.1 and n.sub.2 represent alkylene chains of unknown
length
[0022] Biological Properties:
[0023] Tyrissamycin was evaluated in vitro to determine the minimal
inhibitory concentrations (MIC) against seven gram-positive
bacterial using the microtiter dilution technique. This method is
described by T. B. Conrath, "Handbook of Microtiter Procedures,"
Dynatech Corp., Cambridge, Mass., USA (1972); and T. L. Gavan et al
"Microdilution Test Procedures," in Manual of Clinical
Microbiology, E. H. Lennette, Ed., American Soc. for Microbiol.,
Washington, D.C., USA (1980). These tests are performed in
accordance with procedures established by the National Committee on
Clinical Laboratory Standards (NCCLS). The test procedure is as
follows: Each well of a sterile 96-well microdilution tray is
filled under aseptic conditions with 0.1 ml of Mueller-Hinton
broth. A 0.05-ml sample of the test compound solution is added to a
well in the first row of the tray. A microdilutor apparatus is used
to simultaneously mix the contents of these wells and to transfer
aliquots to each succeeding row of cells to obtain a range of
serially diluted solutions. The last row of wells is untreated and
serves as a control. Each well containing broth and test compound
is inoculated with about ten microliters of inoculum of a given
test microorganism. One well in the last row of wells (which is
free of test compound) is not inoculated and is used as a sterility
control. The trays are sealed and incubated at 37.degree. C. for
16-24 hours. After the incubation period, each plate is evaluated
by determining the lowest concentration of test compound that
visibly inhibits the growth of a given microorganism and recorded
as the minimal inhibitory concentration in .mu.g/ml. The
antimicrobial activity of tyrissamycin is reported in Table 1.
2TABLE 1 Antibacterial Activity of Tyrissamycin Minimal Inhibitory
Microorganism Concentration, .mu.g/ml Streptococcus pneumoniae 4
Streptococcus pyogenes 4 Enterococcus faecalis 32 Enterococcus
faecium 64 Staphylococcus aureus 32 Staphylococcus epidermidis 32
Staphylococcus haemolyticus 32
[0024] As indicated above, the antibiotic of the present invention
is useful as an antimicrobial agent, having utility in inhibiting,
including killing, the growth of microrganisms. It is particularly
useful as an antibacterial agent, especially against gram-positive
bacteria such as those of the genera Streptococcus, Enterococcus
and Staphylococcus.
[0025] The compound may, for example, be used in a method for
treating a host infected by a bacterium or in preventing infection
of said host by said bacterium, comprising administering to said
host tyrissamycin in an amount effective for said prevention or
treatment.
[0026] Hosts include animals, particular mammals such as dogs, cats
and other domestic mammals, and, especially, humans. The dosage
form and mode of administration, as well as the dosage amount, may
be selected by one skilled in the art. The dosage amount will vary
with the severity of the infection, and with the size and species
of the host. Daily dosages for an adult human may be determined by
methods known to one of ordinary skill in the art. Administration
to a mammalian host may, for example, be oral, topical, rectal or
parenteral.
[0027] Pharmaceutical compositions are also provided by the present
invention which comprise tyrissamycin in an amount effective for
the prevention or treatment of infection by a bacterium and a
pharmaceutically acceptable carrier or diluent. The appropriate
solid or liquid vehicle or diluent may be selected, and the
compositions prepared, by methods known to one of ordinary skill in
the art. Examples of such compositions include solid compositions
for oral administration such as solutions, suspensions, syrups or
elixirs and preparations for parenteral administration such as
sterile solutions, suspensions or emulsions. They may also be
manufactured in the form of sterile solid compositions which can be
dissolved in sterile water, physiological saline or some other
suitable sterile injectable medium immediately before use. The
pharmaceutical compositions may contain other antibacterial
agents.
[0028] The compound of the present invention may also be employed
as an antimicrobial agent useful in inhibiting the growth of,
including killing, microorganisms present on a surface or in a
medium outside a living host. The present invention, therefore,
provides a method for inhibiting the growth of bacteria present on
a surface or in a medium, comprising the step of contacting the
surface or medium with tyrissamycin in an amount effective for the
inhibition. Thus, the antibiotic of the present invention may be
employed, for example, in disinfectants for surface treatment, such
as disinfection of surgical instruments, or as preservatives for a
variety of solid and liquid media susceptible to microbial growth.
Suitable amounts of the antibiotic may be determined by methods
known to one of ordinary skill to the art. Compositions comprising
tyrissamycin in an amount effective for inhibiting the growth of
bacteria, and a vehicle or diluent, are also provided by the
present invention.
[0029] The following example is provided for illustrative purposes
only and is not intended to limit the scope of the method.
EXAMPLE 1
Preparation of Tyrissamycin
[0030] General Methods
[0031] Materials
[0032] Hexanes, chloroform, (anhydrous ACS grade), methanol,
acetonitrile (anhydrous HPLC grade) and water were not repurified
or redistilled. Sephadex LH-20 was purchased from Pharmacia LKB,
Uppsala, Sweden.
[0033] Analytical Thin Layer Chromatography (TLC)
[0034] Silica gel precoated thin layer chromatography plates,
Kieselgel 60 F254 on aluminum sheet, 5.times.20 cm, 0.2 mm, were
purchased from EM Separations, Gibbstown, N.J. The plates were
developed in a tank equilibrated with toluene/ethyl acetate (1:2
v/v). The components of the resulting chromatogram were detected
under a UV light, and visualized by phosphomolybidic acid followed
by prolonged heating.
[0035] Preparative TLC
[0036] Silica gel precoated Kieselgel 60 F254 plates on glass,
20.times.20 cm, 2 mm, purchased from EM Separations, were used for
preparative purification. The plates were developed in a tank
equilibrated with toluene/ethyl acetate (1:2 v/v). The components
of the resulting chromatogram were detected under a UV light. The
silica band containing the components was scraped and pressed to a
fine powder, followed by elution with chloroform/methanol (4:1,
v/v). The eluant was then evaporated in vacuo to dryness.
[0037] Analytical HPLC
[0038] The purification of tyrissamycin was monitored by HPLC
analysis on a Microsorb-MV 5 m C-18 column, 4.6 mm i.d..times.25 cm
1. (Rainin Instrumnet Company, Inc., Woburn, Mass.). Analyses were
done on a Hewlett Packard 1090 Liquid Chromatograph, equipped with
a model photodiode array spectrophotometer set at 254 and 280 nm,
and HPLC.sup.3D ChemStation operating software. A gradient solvent
system and 0.01 M potassium phosphate buffer (pH 3.5) was used,
according to the method of D. J. Hook et al (J. Chromatogr. 385,
99, 1987). The eluant was pumped at a flow rate of 1.2 ml/min.
[0039] Analytical Instrumentation
[0040] .sup.1H-NMR and .sup.13C-NMR spectra were obtained on a
Bruker AM-500 500 MHz instrument operating at 500.13 and 125.76
MHz, respectively, using a 5-mm broad-banded probe.
[0041] Extraction and Fermentation of Tyrissamycin
[0042] An aliquot of cryopreserved mycelia from Streptomyces
lividans strain WD15684 is utilized as inoculum for the initial
vegetativestage in medium V13 having the following composition:
Soluble starch, 2%; glucose, 0.5%; NZ-case, 0.3%; yeast extract,
0.2%; fish meat extract, 0.5%; and calcium carbonate, 0.3%. 10
.mu.g/ml of thiostrepton is added aseptically to each flask after
autoclaving to maintain plasmid integrity. The vegetative culture
is incubated at 32.degree. C. and 230 rpm on a rotary shaker for
72-96 hours.
[0043] Following the 72-96 hour incubation, a second set of 500 ml
Erlenmeyer flasks, each containing medium V13 plus 10 .mu.g/ml
thiostrepton, are seeded with 5% of the primary vegetative culture
and shaken at 230 rpm for 3 days at 32.degree. C.
[0044] Said flasks are then pooled to ensure uniformity, and 4% to
5% of the seed culture is aeseptically transferred to production
medium F10A. F10A broth has the following composition: Japanese
soluble starch, 2.5%; dextrose, 0.2%; yeast extract, 0.5%; peptone,
0.5%; calcium carbonate, 0.3%; and distiller's solubles, 0.5%. 10
.mu.g/ml thiostrepton is added post-sterilization.
[0045] The cultures are then fermented on a shaker at 230 rpm for 5
days. On the day of harvest, the contents of all flasks are pooled
into a large vessel, pH and sedimentation readings are performed,
and the batch is forwarded to chemistry. The production of
tyrissamycin reached a maximum of about 30 .mu.g/ml at day 5 in the
production cycle.
[0046] Isolation of Tyrissamycin
[0047] The extraction and fractionation of the above fermentation
broth are monitored by the biological assay described below. Whole
broth (20 liters) is stirred vigorously with ethyl acetate (6
liters) for two hours. The phases are separated and the organic
layer is evaporated in vacuo to dryness. The residue (1.35 g) is
dissolved in 90% aqueous methanol (200 ml) and then partitioned
against hexane (3.times.200 ml). The aqueous methanol layer is
diluted with water to a 65% MeOH solution and partitioned against
pre-equilibrated chloroform (3.times.200 ml). The chloroform layer
is pooled and evaporated to give a crude solid (322 mg). To perform
column chromatography, the solid is loaded to the top of a Sephadex
LH-20 column and eluted with methanol/chloroform (1:1). The eluate
is collected in fractions of 10 ml and monitored for activity in
the biological assay. The combined active eluate (Fraction 13-17,
130 mg) is purified by preparative thin layer chromatography (TLC;
Silica Gel 60 F254, Merck, Toluene-Ethyl acetate 1:2) to yield an
analytically pure sample of tyrissamycin (10 mg).
[0048] Biological Assay of Tyrissamycin
[0049] Assays for detection and evaluation of the antibacterial
activity of tyrissamycin employ various strains of Staphylococcus
aureus. The primary biological assay system consists of S. aureus
strain SA1.
[0050] Culture Growth and Inoculum Production
[0051] SA1 is grown at 37.degree. C. overnight in Antibiotic Assay
Broth (BBL, Cockeysville, Md.). The culture is centrifuged at 4000
rpm for 10 minutes and the medium is decanted. Cells are
resuspended in 0.9% saline. The culture is adjusted to 25%
transmission (600 nm wavelength) using a Coleman Junior
Spectrophotometer. This 25% working cell suspension is stored at
40.degree. C. for use as the assay inoculum.
[0052] Biological Assay Protocol
[0053] All biological assays employ agar diffusion techniques.
Assay agar is Seed Agar (BBL) supplemented after autoclaving with
4% of phosphate-nitrate buffer, pH 7.0, and 0.3% of a 2% solution
of triphenyltetrazolium chloride. For well-agar diffusion assays,
assay agar is seeded with 1% of the working cell suspension of SA1.
The seeded agar is poured into Petri dishes or bioassay trays and
allowed to harden. Wells (7 mm diameter) are cut in the agar, and
40 .mu.l sample aliquots are added to the wells. (For some assays,
3 .mu.l of sample are spotted directly on the surface of the agar).
Plates are incubated at 37.degree. C. for 18-24 hours, and zones of
inhibition (if any) are measured. Inhibition zone sizes correlated
with the activity of tyrissamycin in various samples.
[0054] For bioautographic assays, a thin layer chromatograph (TLC)
plate containing a chromatographed sample is placed in a bioassay
tray. Seeded assay agar is poured over the TLC plate and allowed to
harden. The bioassay tray is incubated at 37.degree. C. for 18-24
hours and examined for the presence of inhibition zones at a
position corresponding to the chemical presence of
tyrissamycin.
[0055] Application of the biological assay to the detection and
isolation of tyrissamycin is illustrated below.
[0056] Monitoring of Bioactivity During Initial Purification of
Tyrissamycin Using a Biological Assay
[0057] Initial purification of tyrissamycin from raw fermentation
broth containing thiostrepton was accomplished using column
chromatographic methods as described above. Biological assays were
performed on Fractions 1-31 resulting from said chromatography.
[0058] These procedures yielded a separation of tyrissamycin from
other biologically active and inactive residues. Results are as
follows:
3 Concentration, Inhibition Sample mg/ml Zone, mm* Fraction 1 1 7.0
Fraction 2 1 9.0 Fraction 3 1 10.0 Fraction 4 1 16.0 Fraction 5 1
20.5 Fraction 6 1 20.5 Fraction 7 1 19.0 Fraction 8 1 15.0 Fraction
9 1 13.0 Fraction 10 1 12.0 Fraction 11 1 11.0 Fraction 12 1 9.0
Fraction 13 (tyrissamycin) 1 9.5 Fraction 14 (tyrissamycin) 1 10.5
Fraction 15 (tyrissamycin) 1 11.0 Fraction 16 (tyrissamycin) 1 10.0
Fraction 17 (tyrissamycin) 1 10.0 Fraction 18 1 9.0 Fraction 19 1
8.0 Fraction 20 1 8.0 Fraction 21 1 8.0 Fraction 22 1 9.0 Fraction
23 1 9.0 Fraction 24 1 8.0 Fraction 25 1 8.0 Fraction 26 1 8.0
Fraction 27 1 7.0 Fraction 28 1 7.0 Fraction 29 1 7.0 Fraction 30 1
7.0 Fraction 31 1 7.0 *Staphylococcus aureus strain SA1; 7 mm agar
wells.
[0059] Monitoring of Bioactivity During Final Purification of
Tyrissamycin Using a Biological Assay:
[0060] Final purification of tyrissamycin from column
chromatographic fractions containing tyrissamycin was achieved as
described above. Biological assays were performed on Fractions 1-4
resulting from said chromatography. These procedures yielded
purified tyrissamycin Results are as follows:
4 Concentration, Inhibition Sample mg/ml Zone, mm* Fraction 1 5 7.0
Fraction 2 5 8.0 Fraction 3 5 8.0 Fraction 4 (tyrissamycin) 5 14.0
*Staphylococcus aureus strain SA1; 7 mm agar wells.
* * * * *