The present invention relates to novel antibiotics. In particular,
this invention relates to antibiotic stalobacins H and I produced by
sp. PBJ-5360-STR-1-21, the microorganism which produces the antibiotics,
and a process for producing the same.
It is well known that antibacterial activity of a particular antibiotic
varies depending on a nature of bacteria to be treated and the effect of the antibiotic
often reduces because of the advent of resistant strains. The advent of multiple
drug resistant bacteria has recently become a big problem. Accordingly, development
of novel and effective antibiotics has been desired for performing effective treatments.
Above all, many Gram-positive bacteria, such as Staphylococcus, hemolytic
streptococcus or the like are resistant to antibiotics, and there is continuous
need for the development of novel antibiotics having high potency to these Gram-positive
The present invention provides antibiotic stalobacins selected from
the group consisting of antibiotic stalobacins H and I produced by the above-noted
Pseudomonus sp. PBJ-5360-STR-1-21. These antibiotics are peptide antibiotics
which are produced by said Pseudomonas sp. PBJ-5360-STR-1-21. Stalobacins
H and I (hereinafter sometimes referred to as merely "stalobacins") are obtained
in the form of a mixture of closely related analogs by cultivating said
Their antibacterial activities are much more potent than those
of known antibiotics. Stalobacins H and I have physico-chemical properties as shown
in the following Table 1.
Physico-chemical Properties of Stalobacins H and I:
* Column: Develosil 5C18, 4.6 i.d. x 250 mm
m.p. (°C) (as Na salt)
molecular ion (m/z)
IR (KBr) (cm-1)
1654, 1597, 1525
1651, 1596, 1527
Retention time (min.)
Amino Acid Analysis
Mobile phase: CH3CN/2 mM H3PO4 (containing 50 mM
Na2SO4) = 43/57
Flow rate: 1 ml/min.
1) Hydroxyaspartic acid
The antibiotic stalobacins of the present invention characterized
by the above properties have been found to have excellent antibacterial activities
and in vivo, showing potent effects especially on Gram-positive
Thus, the antibiotic stalobacins of the present invention show excellent
activities, and have higher activities especially against Gram-positive bacteria,
as shown below in Table 2.
No death was observed in acute toxity test by intravenous administration
of 300 mg/kg and 500 mg/kg of the antibiotic stalobacins to mice.
Antibiotic stalobacins H and I of the present invention are produced
by cultivating Pseudomonas sp. PBJ-5360-STR-1-21, a variant derived from
Pseudomonas sp. PBJ-5360 (BIKOKEN deposition No. 10578, FERM BP-4342),
which produces a mixture of stalobacins, aerobically in a liquid medium containing
assimilable carbon sources, nitrogen sources and mineral salts in a conventional
manner. This bacterium has been identified as the above mentioned strain by cultivating
it according to the method as hereinafter described in Experiment 2 and examining
comprehensively its morphology, culture properties, physiological and biochemical
properties in reference to the description of Bergey's Manual of Systematic Bacteriology,
Vol. 1 (1984). This strain may undergo a spontaneous or artificial mutation, and
it is obvious to a person skilled in the art that such mutants should be included
in the scope of the present invention, as far as they retain an ability to produce
the stalobacins of the present invention. Thus, the present invention provides
also Pseudomonas sp. PBJ-5360-STR-1-21 producing novel antibiotic stalobacins
H or I and mutants thereof having an ability to produce said antibiotic stalobacins.
Pseudomonas sp. PBJ-5360-STR-1-21 was deposited under accession No. FERM
P-14149 with National Institute of Bioscience and Human Technology, Higashi 1-1-3,
Tsukubashi, Ibaraki Pref. Japan, on February 16, 1994, and said original deposit
was transferred to International deposition under Budapest Treaty on April 28,
1994, and given Accession number FERM BP-4661.
Furthermore, the present invention provides a process for producing
antibiotic stalobacins H and I by cultivating such strain.
Ordinary compositions of medium and ordinary conditions used for
conventional cultivation for producing antibiotics can be adopted. In principle,
the medium includes carbon sources, nitrogen sources, mineral salts and the like.
If necessary, vitamins, precursors or the like can be added. Examples of carbon
sources are glucose, starch, dextrin, glycerin, molasses, organic acids and the
like, and these carbon sources may be used alone or in a mixture thereof. Examples
of nitrogen sources are soybean powder, corn steep liquor, meat extract, yeast
extract, cotton seed powder, peptone, wheat embryos, ammonium sulfate, ammonium
nitrate and the like, and these nitrogen sources may be used alone or in a mixture
thereof. Examples of mineral salts are calcium carbonate, sodium chloride, potassium
chloride, magnesium sulfate, cupric sulfate, manganese chloride, zinc sulfate,
cobalt chloride, various phosphates and the like. These mineral salts may be added
to a medium when required. A sufficient amount of antibiotic stalobacins H and
I is produced by cultivating Pseudomonas sp. PBJ-5360-STR-1-21 of the present
invention in an appropriate medium at temperatures from 20 to 35°C, preferably
25 to 29°C, for about 1 to 7 days. The product is then isolated and purified,
if necessary, from the culture in a conventional manner. All of such procedures
are well known to a person skilled in the art.
The antibiotic stalobacins of the present invention are believed
to be useful for treating various infections, in particular, treating infections
caused by multiple drug-resistant Gram-positive bacteria, since they exhibit marked
antibacterial activities in vivo and in vitro.
Figure 1 is a graph showing IR spectrum of stalobacin H.
Figure 2 is a graph showing IR spectrum of stalobacin I.
Figure 3 is a graph showing NMR spectrum of stalobacin H.
Figure 4 is a graph showing MMR spectrum of stalobacin I.
The present invention will be explained in more detail below by illustrating
Examples and Experiments.
(a) Fermentation Step:
Eight hundred ml of a medium (adjusted to pH 7 with 2N-NaOH) consisting
of 1.0% glucose, 0.5% yeast extract (Difco) and tap water in a 2 L Erlenmeyer flask
was inoculated with a seed strain of Pseudomonas sp. PBJ-5360-STR-1-21
(kept at -80°C in a 2 ml vial), and the resultant mixture was subjected to a shaking
cultivation at 180 rpm with 70 mm of shaking breadth at 28°C for 22 hours. The
culture (800 ml) was inoculated to 20 L of a medium (adjusted to pH 7 with 2N-NaOH)
containing 1.0% glucose, 0.4% yeast extract (Difco), 1.0% malt extract (Difco),
0.1% polypeptone (Nippon Seiyaku) and tap water in a 30 L jar fermenter, and the
resultant mixture was cultivated with agitation at 200 rpm, with 14 L/min of aeration
under 0.35 kg/cm2G of inner pressure, at 28°C for 21 hours.
Then, 8 L of this culture was inoculated to 125 L of a medium (adjusted
to pH 7 with 2N-NaOH) consisting of 2.0% soluble starch, 2.0% powdery yeast, 1.5%
β-cyclodextrin, 0.5% olive oil, 0.3% magnesium chloride•6H2O,
0.1% potassium dihydrogenphosphate, 0.0008% antifoaming reagent ADECANOL (Asahi
Denka Kogyo) LG109 and tap water in a 250 L tank, and the resultant mixture was
cultivated with 65 L/min of aeration, under 0.35 kg/cm2G of inner pressure,
with agitation at 350 rpm at 28°C for 72 hours.
(b) Separation Step:
To 138 L of the culture obtained in the foregoing step was added
1.4 L of chloroform for sterilization. Then, 15 L of Amberlite XAD-7 (Organo)
was added and the resulting mixture was mixed with stirring for 3 hours for a batch
adsorption of the active substances onto the resin. The resin was recovered using
a #140 mesh stainless steel sieve. The resin was washed with water, put in a glass
column (inner diameter: 20 cm), washed with 40 L of water, 40 L of 30% methanol
and then 15 L of 50% methanol in 20 mM phosphate buffer (pH 7.5), and the active
substances were eluted by 40 L of 60% methanol in 20 mM phosphate buffer (pH 7.5).
Fractions (20 L) showing antibacterial activity to S. aureus
JC-1 were collected, concentrated in vacuo
to 3 L. The concentrate was washed
with 3 L of ethyl acetate to remove lipophilic materials. The ethyl acetate contained
in the aqueous layer was evaporated in vacuo. The active substances were
adsorpted on Amberlite XAD-7 (Organo) in a 1 L column (inner diameter 6.5 cm)
and the resin was washed with 2 L of water. The elution was carried out using 2
L of 30% aqueous MeOH and 3 L of 50% aqueous MeOH. The eluted fractions were subjected
to a HPLC analysis, and the fractions containing stalobacins were collected, adjusted
to pH 7.0 with 2N-HCl, concentrated in vacuo and lyophilized to obtain 3890
mg of powder.
(c) Purification Step:
The First Purification Step:
One thousand and eight hundred twenty mg of the crude powder obtained
in the foregoing step was dissolved in 60 ml of 20 mM phosphate buffer (pH 7.5).
The solution was subjected to a preparative high-speed liquid chromatography using
YMC ODS column [S-15/30 µ, 5.0 x 50 cm, eluent: acetonitrile/20 mM phosphate buffer
(pH 7.5). 50 mM sodium sulfate = 40/60, flow rate: 50 ml/min, UV detection; 210
nm] to obtain a fraction (2.4 L) containing stalobacins H and I as main ingredients.
The acetonitrile in the fraction was distilled off and the residue was passed
through a column of Diaion HP-20 (Mitsubishi Kasei Corporation). The resin was
washed with water, and the adsorbed components were eluted with 60% aqueous acetone.
The acetone in the eluate was distilled away in vacuo and the residue was
lyophilized to obtain 126 mg of powder.
The Second Purification Step:
The powder obtained in the above step was purified by preparative
high-speed liquid chromatography under the following conditions to obtain stalobacins
H and I.
The powder (126 mg) was dissolved in 7 ml of 50 mM phosphate buffer
(pH 7.0). Fifteen mg of the sample per one procedure was charged into Asahipak
ODP-90 column (inner diameter 21.5 mm x 300 mm, eluent: acetonitrile solution
of 20 mM AcOH/aqueous solution of 20 mM AcOH = 40/60, flow rate: 8 ml/min, UV detection:
220 nm), and stalobacin H was collected from the fractions of 144 ml to 184 ml
and stalobacin I was collected from the fractions of 216 ml to 288 ml. This fractionation
was repeated, and collected fractions were neutralized to pH 7.0 with aqueous
1N-NaOH. The acetonitrile was distilled off in vacuo and NaCl was added
to the residue to obtain 5% NaCl concentration. The resultant mixture was adjusted
to pH 7.5 with 1N NaOH and passed through MCI GEL CHP20P column (75 to 150 µ, Mitsubishi
Kasei) which had been equilibrated with 5% aqueous NaCl solution. The column was
washed with water and eluted with 70% aqueous MeOH. The MeOH in the eluate was
distilled off in vacuo
and the residue was lyophilized to give 12 mg of pure
stalobacin H and 38 mg of pure stalobacin I.
Physico-chemical properties of stalobacins H and I obtained in Example
1 are shown in Table 2. IR spectra of stalobacins H and I were shown in Figs. 1
and 2 respectively, and NMR spectra of stalobacins H and I were shown in Figs.
3 and 4 respectively.
Experiments 1 Antibacterial Activity in vitro and
1) In vitro Antibacterial Activity:
Antibacterial activity in vitro of antibiotic stalobacins
H and I obtained in Example 1 was assayed by the agar dilution method. The results
are shown in Table 2.
2) In vivo Antibacterial Activity:
Stalobacin (µg/ml), 106 cfu/ml)
S. aureus FDA JC-1
S. faecalis SR1004
S. aureus 3626 (MRSA)
Antibacterial activity in vivo of stalobacin I was assayed.
Mice were intraperitoneally challenged with infectious bacteria. One hour after
the challenge, the test compound was subcutaneously administered. ED50
was calculated on the basis of survival ratio on 7th day after the challenge. MIC
was determined according to the agar dilution method. The results are shown in
Experiment 2 Bacteriological Properties of PBJ-5360 and PBJ-5360-STR-1-21:
Protective Effect of Stalobacin I in Mice Systemically Infected:
S. aureus SR3637 (H-MRSA)
S. pneumoniae Type I
E. faecalis SR1004
PBJ-5360-STR-1-21 of the present invention was obtained as a mutant
of the above-mentioned PBJ-5360 strain. PBJ-5360 was isolated from the soil collected
in Kyoto, Japan. Various bacteriological properties of PBJ-5360-STR-1-21 of the
present invention are shown below. Cultivation was effected at 28°C in principle.
It is a Gram-negative rod. Its size is 0.3 - 0.5 (µm) x 0.8 - 1.3
(µm). It vigorously moves with one or more polar flagella.
B. Characteristics of Culture
1) Cultivation in meat infusion medium:
Growth of the bacteria was hardly observed. Off white translucent
precipitates formed very slightly at the bottom of the test tube.
2) Meat infusion agar stab culture:
Growth in thread form or small nipple form along the stab line was
observed. Neither evolution of gas nor production of pigment was observed. Reddish
thin bacterial plaque appeared on the surface, but this bacterial plaque became
translucent and light brown with the lapse of time and verrucose projections were
observed in several places. It is an aerobic bacterium.
3) Meat infusion agar slant culture:
The growth of the bacteria was not so rapid and began at 28°C after
two days (observed with naked eyes). The bacteria grew in thread form, and its
bacterial plaque was translucent and light yellow with flat swelling having spotty
appearance. The periphery was whole peripheral. Then, the bacterial plaque grew
favorably in thread form or verrucose form with gloss. Thus, a wet slightly reddished
translucent brown bacterial plaque was obtained. The periphery was wavy or long
wavy. Production of any gas or pigment was not observed.
4) Meat infusion gelatin stab culture:
Cultivation was effected at room temperature (22 - 25°C). The gelatin
was slightly liquefied.
5) Cultivation on the meat infusion agar plane medium:
The growth of the bacteria was not so rapid. The colony became visible
at 28°C after two days. The colony was initially small, spotty, translucent and
brown with a whole periphery. The colony was two small to be observed about its
swelling. Then, the colony grew in a spotty or circular form and with whole periphery
and the swelling was flat or convex circular. The colony was translucent and brown
with gloss. Neither gas nor soluble pigment was produced.
6) Characteristics in litmus milk culture:
An acid formation did not occur, and peptonization occurred but the
reaction began after 14 days. Thus, the reaction was rather slowly. No gas evolved.
C. Physiological and Biochemical Properties
- Catalase test: positive
- Oxidase test: positive
- OF-test: negative (showing to be alkaline)
- Hemolytic test: positive (weakly)
- Viability at 5°C: negative
- Production of H2S: negative
- Nitrate reduction: positive
- Denitrification: negative (although no nitrogen gas was evolved, it seemed
to reduce No2-)
- Citrate utilization: negative (Christensen medium and Simons medium)
- Growth on NAC agar medium: negative (nonviable)
- Production of indole: negative
- Voges-Proskauer reaction (Voges-Proskauer test):
- Methyl Red test: negative
- Arginine dihydrolase test: weakly positive
- Lysine decarboxylase test: positive
- Ornithine decarboxylase test: positive
- Esculin hydrolysis: negative
- DNase test: negative
- Starch hydrolysis: negative
- ONPG test (cultivated at 37°C): negative
- Acylamidase test: positive
- Phosphatase test: positive
- Chitin hydrolysis: negative
- Productivity of levan from sucrose: positive
- Productivity of acids and gas from sugars:
Neither acids nor gases were produced from the following 13 sugars: glucose, fructose,
galactose, mannose, xylose, arabinose, maltose, lactose, rhamnose, sucrose, cellobiose,
trehalose and mannitol.
- Accumulation of poly-β-hydroxybutyrate in the cell: negative
- Utilization of carbon sources:
On the medium containing minerals, glucose and calcium 2-keto-gluconate can be
used as a sole carbon source for the formation of the cells. In this case, it
seemed that specific vitamins for growth were not required. On the other hand,
D-(+)-trehalose, DL-arginine, geraniol, β-alanine, L-valine and inositol
were not be utilized.
- G+C mole % (HPLC method): 60.4% (A+T mole % = 39.6%)
In view of the above test results, PBJ-5360-STR-1-21 is an aerobic
Gram-negative rod and moves actively in a liquid medium using one or more polar
flagella. It is positive for catalase and contains oxidase. It was negative for
OF-test (showing to be alkaline). In view of these observations, it is apparent
that the present bacterium belongs to the genus Pseudomonas in Family
When the inventors compared the above properties with those of the
bacterial complexes which are incapable of accumulating poly-β-hydroxybutyrate
(PHB) in their cells, which complexes are described in Bergey's Manual of Systematic
Bacteriology, Vol. 1 (1984) on Genus Pseudomonas, the present inventors
failed to find any bacterium having those properties consistent or analogous to
the properties described above. This bacterium appears a considerably unusual strain
of Pseudomonas because it hydrolyzes arginine and decarboxylates lysine
and ornithine. The G+C mole % value of 60.4% indicates that the strain belongs
to a group having lower G+C value in Pseudomonas. Thus, in view of the
various properties mentioned above, the present bacterium has been identified as
Pseudomonas sp. PBJ-5360-STR-1-21. These properties were consistent with
those of the parent strain PBJ-5360.