Neo-nocardin, a New Antibiotic Produced by a
New Species of Nocardia.

1. Taxonomical Studies of the Organism, Production
and Extraction of the Antibiotic and its
Antibacterial Activities.
By
Ichiro UESAKA *J::m ~ ll~(Received April 4, 1951)

Introduction.

Though there have been reported many antibiotics produced by strepto.
myces, reports on the antibiotic activities of nocardia, closely related genus
of streptomyces, are comparatively rare (1)(2).
During my searches for new antibiotics produced by actinomycetes, I
have found a new species of nocardia which produced a new antibiotic designated as neo-nocardin,
This paper describes the taxonomical studies of this strain, productionof the active substance by the surface culture, antibacterial activity of thefermented broth, method of extraction, antibacterial activity and toxicity ofcrude extract, and finally the antagonisitic activity of the crude extract on
tubercle bacillus in vitro.

1. Taxonomical Studies
In my collection of actinornycetes from soils I have found a strain of nocardia which showed a marked antibacter lal activity and this strain was designated as strain No. A. 422.
*From the Bacter'iological Laboratory (Chief:

Prof. Saburo UYEDA) of theTuberculosi s Research Institute, Kyoto University

22lchiro UESAKAa). Morphological Studies
For the continuous obser vat ion of the organism the hanging drop method
was used.
Modified CZAPEK'S agar or solution was used for the culture medium.
Lactose was used for the carbon source in this medium, because
lactose was superior to other sugars for the separatlon and dispersion ofmycelia,1) Observation of fresh preparationsFirst day of cultivation (Fig. 1) : Mycelia are slightly curved.
Cells are double-refracted.
Usually
two germ-tubes, about 0.8 to 18 microns in length.
Second (Fig. 2) and third (Fig 3) day of cultivation:
Mycelia grow further, branch abundantly and twine
each other.
Sometimes granules become visible in
3 days old culture.

Fig. 1.

Fig. 2

Fig. 3

c

A

,

~

l

B

~

Fig. IV~.oO

Fifth and sixth day of cultivation and thereafter (Fig.
4) : Mycelia cling together andthen separate into various
forms of cells and disperse in
the medium.
In these cells
double-refracted granules arepresent.

~1;~

oCP D
•

Neo-nocardin, a New Antibiotic

232) Obsevation of stained preparations
Methylene blue stain:
Preparation of the 5th day culture was stainedwith methylene blue (Fig. 5).
It was demonstrated that the mycelia in Fig. 4
were really made up of chainsof rods or branched rods asshown in Fig. 5 (A) an.d (D).
In the middle of these rods there exist condensed mater ialscAstained dee p blue.
Separated
cells, about 0.8 by 1.3 microns,
we r-e also sta inc d deep blue asshown in Fig. 5 (B).
Acid-fast stain:
Thoughthe mycelium was weak acidFig. 5fast, cells and their prestadium ... Fig. 5 (A), (C) and (D) .. were non acid-fast.
Gram stain:
Gram-positive.

rbl

Physiological studies

Separated cells were sown on various media and incubated at 37°C in orderto establish a spectrum of nutrient sources and to observe the effects on certain different media
The results are so comprehensive that only the cha racter istic properties will be described.
(1) Glucose agar:
Abundant growth, at first yellowish brown, thengradually reddish brown.
Aerial mycelium scant, white, at the margin ofcolonies.
Red wine colored pigment.
(2) Synthetic agar:
Pinkish white, punctiform growth.
Grayish pink
pigment.
(3) KRAINSKY'S agar:

Thin,pale yellow colonies,

partly white puncti-

form aerial mycelium,
Yellow pigment.
(4) Gelatin stab:
Yellowish brown colony grows sinking in the medium,but no growth along the line of puncture.
No aer ial mycelium.
Yellowish
browh pigment.
No liquefaction.
(5) Skimmed milk:
No coagulation.
Liquefaction after twe days.
Brown pigment in the bottom or tube.
The reaction gradually becomes
alkaline.
(6) Blood agar:
Medium becomes black.

Numerous minute colonies.
No hemolysis.

No aerial mycelium.

(7) Egg media:
Red colonies all over the surface of the medium.
Grayaerial mycelium at the under margin of colonies.
Slightly grayish pinkIchiro KAAfISAKA

24---~--~-----------

pigment.
(8) Starch is hydrolyzed,(9) Nitrates are not reduced.
(10) Tyrosinase is not present.
As the nitrogen sources peptone and(11) Nitrogen and carbon sources:
sodium nitrate are suitable.
Galactose, mannose, lactose, maltose, dextrin,
inulin, starch, glycogen, mannitol, and sorbitol stimulate the growth of theorganism as the carbon sources.

(c) Conclusion of the taxonomical studies.
Morphological characteristics of No. 422 strain on the synthetic media(lactose as the carbon source) are as follows:
In the early stages of growth
this strain forms a long mycelium; but, after 5 to 6 days, the filaments
break up into various forms of fragments, then into cylindrical short cells
and I have never seen conidia in chains.
Mycelia are weak acid-fast, but
the cells and the fragments of mycelia are not acid-fast.
From these characteristics this organism must belong to Nocardia according to the new nomenclature by WAKSMAN and HENRICI(3).
I could not find out, however, thespecies identical with this organism in the 33 species of genus Nocardia.
Since this organism seems to belong to new species, I would like to namethis strain as Nocardia Kuroishi n. sp..

2. Production of the Active Substance by the Surface Culture(a)Factors influencing the antibacterial activity of culture fluid of A. 422

strain.
Sodium chloride suppressed the activity of the culture fluid.
The inhibition of activity by salt increased with the increase in the concentration ofsalt.
The activity of culture fluid was also found to decrease with the deerease of pH.
The rabbit serum did not apparently influenced the activity.
From these results it was found that the simplified broth (0.5 per cent meat
extract and 0.5 per cent peptone, pH 7) waseuitable for the assay of antibacterial properties of the culture fluid of this strain.
(b) Cultural conditions affecting the production of antibiotic substance(neo-nocardin).
(1) Addition of agar in the culture media:
Addition of 0.2 per cent or
0.3 per cent agar in the glucose broth stimulated the growth and pigment
production of this organism and increased its antibacterial properties.
(2) Intial pH of the culture me dta :The bacteriostatic effect of theculture fluid was investigated at the initial pH of 6.4 to 8.2.
Tqe maximum
production of the antibiotic was obtained at initial pH of 7.0 to 7.6 and theactivity rapidly decreased when the pH increased to over 7.8.

Neo-nocardin, a New Antibiotic- - - - - - - ---------- -----------(3) Effect of ingredients of culture media on the antibacterial properties:
Various carbon and nitrogen sources were investigated.
As the carbon
source glucose and starch have the same pr opcr ty for the prduction of nconocardin.
Though there was no difference in the maximum yield of antibiotic, glycerol had the property to produce nco-nocardin earlier than glucose.
Peptone was superior to sodium nitrate as the nitrogen source.

3.

Antibacterial Properties of the Fermented BrothThe fermented broth was tested for the antibacterial activity against various bacteria (Table 1).
The fermented broth inhibited the growth of gramTable 1

Bacteriostatic activity of the fermented broth of No. A. 422
against various bacteria
Bacteriostatic activity
at dilution of

Staphylococcus aur eus T'er aji rn n
Vibrio cholerae Inabn

1: 160,000
1:
3,200

Escherichia coli
Salmonella paratyphi A

1:
1:

3,200
::,2CO

Salmonella par aty phi B
Eberthella ty pho sa Boxhill

1:
1:
1:
1:
1:
1:
1:

100
1,600
1,600

Eberthella ty pho sa H 901
Shigella dysenteriae Hanabusa
Shigella parady senter iae Komagome B
Bacillus anth rac is
Bacillus subtilis
Pseudomonas ae rug ino sa

< 1:

1,600
1CO

1,600
100
10

positive and gr arn-negative bacteria.
The difference of suscept ibil ity among
the different strains in the same species of the test organisms is widely recognized in the case of penicillin and other antibiotics.

This phenomenon was

also observed in neo-nocar din (Table 2).
Table 2Difference of susceptibility among the different strains of staphylococci
Strains

Species

Penicillin
su seep tibili ty

Susceptibility to
the culture fluidof No. A. 422

Wood

13.3~~

T'erajirn a

Yamase16.3
17.9

1: 64,000
1: 40,000
1: 8,000

Heatley

16.3

1.

Hodo

20.4

Kimura 1

12.1

1:

I Noguchi

7.9

1: 64,OCO--citre-~---r-Shi~~ 3

27.5

1:

aureus2,COO

1: 1,000100

1 - - - - - - - - - - - -- -------~..---- --c

albus

*

2,COO

I

Numbers represent the length of inhibition zones in mm tested by the cup me thod.

26

Ichiro UESAKA

4.

Method of Extraction(a) Stability ox the active substance in the fermented br oth :The fermented broth was adjusted to various pH and heated at 100°C.
Any loss of potency was not observed after 10 to 30 minutes prescrvation :after 60 minutes preservat.ion, however, the potency decreased less than 1/10
compared with that of the original broth.
On the contrary, the potency
increased after 20 minutes preservation and such increase in potency was also
observed when Eberthella ty phosa or Escherichia coli was used in place ofStaphylococcus aureus as the test organism.
(b) The active substance in the fermented broth was hardly dissolved in thesolvents tested.
(c) Changes of antibacterial activity by the removal of mycelium:
For the extraction of the active substance from the fermented broth it isnecessary to remove mycelium and agar from the broth.
The antibacterial activity of the filtrated broth was remarkably decreasedby the removal of mycelium and agar independently of the methods of removal
whether they were removed by centrifugation or by filtration with gauze.
No decrease in potency was observed, however, when mycelium and agar
were removed after the broth had been preserved at Hxrc for 20 minutes.
(d) Adsorption of the active substance from the broth filtrate to carbon or
to diatomaceous earth:
1) To activated carbon
After the culture filtrate was adjusted to various pH with HCl or NaOH
and then treated with 1 per cent activated carbon and centrifuged, the potency of the supernatant was tested.
Considerable amount of the active substance remained in the supernatant between pH 2 and 4.
Adsorption of tho
active substance to the activated carbon. was perfect between pH 5 and 7,
while at pH 8 a small amount of the active substance remained in the supernatant.
2) To diatomaceous earth
The greatest amount of the active substance remained in the supernatant
at pH 2.
For this reason the broth filtrate wrs adjusted with HCI to pH 2
and treated with various concentrations of diatomaceous earth and centrifuged ; the potency of the supernatant was tested.
The decrease of potency
in supernatant was not observed by the treatment of 0.1 per cent diatomaceous
earth, and mycelium and agar were entirely eliminated from the supernatant.
(e) Elution of the active substance from carbon
Elution into water, absolute ethanol or absolute methanol from the acti-

27

Neo-nocardin, a New Antibioticvated carbon was unsatisfactory,
Elution into ethanol with or without Hel
was then tested.
The greatest amount of the active substance was eluted
into 0.1 N HCI ethanol.
Elution into 0.1 N HCI ethanol was, therefore, tcsted in compar ison with elution into methanol with or without HCl.
The
greatest yield was gained by 0.1 N HCI methanol and the next was by 4/100
N HCI methanol and it was recognized that HCI ethanol was in general not agood eluent compared with Hel methanol.
Among HCI methanols 4/100 N HCl
methanol was superior to 0.1 N HCI methanol for the formation of precipitateby adding ether into the eluent containing the active substance.

Table 3

Adsorption of neo-riocard in to the diatomaceous earth

1)

Diatomaceous earth

1%

I

Colorofdepth

i

fluid

II'

I

I Antistaphylococcal
I

activity--"--------1

Original broth (pH 8)+11

1 : 32,000

After the preservation at
IGO°C for 20 minutes+11

1 : 64,COO

+2

1:

2,000

+3

1:

100

-+- "{

1:

100

+(1

1:

100

Supernatant after thead sorption to thediatomaceous earth
from the heated
broth

2)
Diatomaceous earth
0.5.%

Antistaphylococcal
act.ivi ty

Original broth

1: 64,000

After the preservation at lCO°C for 20 min

1: 128,OCO

Supernatant aft.er the adsorption to thediatomaceous earth from the heatedbroth at pH 2.

1:

Sl,2003)

Diatomaceous earth
O.2;?~

Anti staphylococcal
activity

Original brot.h

1: 8000

After the preservation at 100"C for 20 min.
Supernatant ai te r the adsorption to thediatomaceous earth from the heatedbroth at pH 2

1: 8000

1: 4000

lchiro UESAKA.

28

4)
Diatomaceous earth
0.2%

Antistaphylococcal
activity

Original broth
After the preservation at 100°C for 20 min.
Supernatant after the adsorption to thediatomaceous earth from the heatedbroth at pH 2.

1: 4000
1: 8000

1: 8000

From the above-mentioned results the whole process of extracting the active substance (neo-nocardin) from the broth to the crude hydrochloride can besummarized as Table 4.
Table 4 Extraction of neonocardin from the fermented brothFermented broth----~--_.-

preserved at 100°C for 20 min., cooled, then filtered with gauze~

Filtrate
pH 2 with HCI, added with O.1~6 diatomaceous earth, centrifuged
at 2500 r. p. m. for 20 to 30 min.
~Supernatant~

pH 5 -- 6 with NaOH, added with 1% activated carbon, filtered.
~++

Sediment

FiltratecolorlessI'" water 010 volume of the broth)
Washed with~

Washed with methanol (5 ml/g)~

Washed with ether+

Eluted into ~oo N HCI methanol more than 3 times, centrifuged1-------------------,~
Eluate~Sediment~

precipitated with ether (3 -- 4 volumes of eluate)~

dissolved into absolute methanol, centrifuged1-------,
1~

Supernatant~

Sediment
Remains of carbon

Precipitated with ether (3 -- 4 volumed of eluate)1

Crude neo-nocardin

The neo-noardin hydrochloride is hygroscopic, yellowish gray to grayish
white powder.

Neo-nocardin, a New Antibiotic

29

5. Antibacterial activity and toxicity of crude neo-nocardin hydrochloride(a) Antibacterial activity; The antibacterial activity of crude neo-nocardinhydrochloride was tested by the dilution method.
The results are shown in
Table 5.
The antibacterial activity varied more or less with samples.
Table 5

Antibacterial activity of crude neo-nocard in

I Antibacterial activity

Test organism

1: 6,400,000
1: 1,600,000
1: 800,000
1: 800,000
1: 800,000
1: 400,000
1: 100,000

Staphylococcus aureus Terajima
Vibrio cholerae Inaba
Eberthella typhosa B::>xhill
Eberthella typhosa H 901
Shigella dysen teriae Hanabusa
Bacillus anthracis
Bacillus subtilis

Test medium: 0.5% Meat Extract + 0.5% Peptone(b) Toxicity in mice:
The crude neo-nocardin hydrochloride of 0.1, 0.5, 1.0
and 2.0 mg were dissolved in 0.5 ml of physiological NaCI solution and each
0.5 ml was injected into mice intraper itoneally (5 mice per one kind of concentration).
They were observed for two weeks and all animals survived within'these dosis of the antibiotic and any remarkable toxic effects were not observed.

6. Antagonistic Activity of Crude N eo-nocardin Hydrochloride on Tubercle Bacilli In Vitro
The antagonistic activity of crude neo-nocardin hydrochloride on tubercle
bacilli was investigated.
It is widely known there are antibiotics of which
activities decrease when these are preserved at 37°C.
As seen in Fig. 6, though the activities ot streptomycin and neo-nocardin
Fig. 6 Changes of antibiotic activities of neo-nocardin and streptomycin on
tubercle bacilli by:the preservation at 37°C-

1>-. I: ,o"'r~:::::~~.... ... -~o... -..,. .......

__

'

~

....

, ...,~_

Strcptomy:i 11.......

' ...., .....J.r------- Ne Oo-in 0 Ca. Y d.I 11

~

,}

.

~
(/)

o

.....

' .... ....

C>«
::$

U
~

Cl)

..0

F

14

22

30

38

__________~)

Ti me l d.CLI'-S )Ichiro UESAKA

30

on tubercle bacilli in Kirchner media were the same after 14 days preservation,
their activities gradually decrease with progressing time.
But the decreaseof activity of neo-nocardin was more rapid than that of streptomycin and after
45 days incubation the activity of the former was only about 1/20 as comparedwith that of the latter.
From the above-mentioned results is it reasonable
to consider that neo-nocardin might also be ineffective in vivo?
I neverthink so.
Drugs are as a rule administered everyday for patients or for experimentally infected animals so far as any toxic effect is not found.
It is usually outof question whether the activity ot drug might be decreased at the next dayof administration or not.
The drug should maintain its activity at least
during the day of administration, and on the next day the growth of thebacilli may be inhibited by the administration of the day.
In this reason
I think it is worthless to determine the activity of drugs after 5 or 6 weeks
incubation as it is usually practised by the antituberculous agents.
I think
it is necessary to devise new methods which arc able to determine the activity
at least a few days after incubation.
According to this principle I havedevised two methods of determining the activities of antituberculous agentsin a few days, and using these methods I have cpmpared the activity of
neo-nocardin on tubercle bacilli with that of streptomycin.
(a) Microscopic method:
The agents'" streptomycin and crude neo-nocardin ... dissolved in distilled water.
Subsequent dilutions were made directly
into 1.0 ml of Kirchner media.
Each tubes were inoculated with saline
suspension ot tubercle bacilli.
The control tubes containing no agents but
inoculated with tubercle bacilli were prepared.
All these tubes were incubated at 3TC.
From the next day on one control tube a day was taken out
from the incubator and the supernatant was removed without agitating thecontent.
The remaining 0.2 to 0.3 ml of fluid was soaked up and dropped on
several pieces of slide glasses.
These glasses were dried, fixed and stainedin due form and then examined microscopically.
On this occasion I examinedat first by dry system (80 X) and if. the bacterial growth was not· found I
examined by oil immersion system (800 X).
If the initial growth of thebacilli, serpentine growth specific to this organism, was recognized, I treated
Table 6 Microscopic method1)

Dry system (80X Magnification, after 7 days)
Dilutions l

An tibiotics

J

2

4

8

16

32

6,1

128

256

512

1024 2018

X

lO·t I Control",---------------------------- ----I

Streptomycin
Neo-nocardin

-

+

+

+

+

+

+

+

z

(b

0

~

C/l

~

'"1

~

(b

=
S
o
0

III
'"1

0..

5'

'0

0.:

'0...1

rJJ

g

3

'<:
~

S'

~

s=
::so'

Table 7 Subcult.ivation method.
X 104

Subcultured onegg media after

I2

4

8

16 32

day (s)lStreptomYcin
1
Neo-nocardinf---3

lStreptomYcin__ ~e~-nocardin

256

1024

512

2048

78

68

59

48

38 46 29 39 46

83

63

79

45

3

---

-

1 ca. 130 ca. 130

111

Ei1

ca. 120 .

ca. 140ca. 120II

ca. 120ca. 130

I

I

00

82

ca. 130 ca. 140 ca. 140

ca.2oo

I3

8

89

78

ca. 120 ca. 150 ca. 150

II ca.150 I

2

3

1

3

-----

C

35

ca. 200 ca.200 >200

>200

ca. 200 >200

>200

5

0

Neo-nocardin

5

12 17 48 40: ca. 200 ca. 150 ca. 200 >200..........................................

'"1
rJJ

....0
rJJ

'<:

85

ftreptomYcinl\:),;::..

18
--

I

rJJ

'(l)"'"

3--_._'._---,",-~-_._-.--

....

3
3(b
::l

I6 1 1 0 4
22 33 55 22

rJJ

I

18 17 5012-----_._--~

3

128

64

~

I Co~troI I Co~trol-_.'~'--"'-

N

0'
'"'"
r;'

I

~

I

I

0)

I

I

N

I

I

,;::..

Coil

~
:x

c(")

~

~

III

1

e.....

O'l

r;'

f-l

III

N

I

'"'"
o'

00

l:l

Note: (1) 0.04 ml of saline suspension of tubercle bacilli (0.05 mg/ml) was inoculated into each tubes

I

N

III

UI

I

Ol

of Kirchner media containing serially diluted antibiotics and after 1, 3 and 5 days of

I-h
~

(l)

'"1

incubation the bacilli were subcultivated on egg media.
(2) Control 1 is the one which was subcultivated from tubes containing no antibiotics. Control 2 is the one which was subcultivated from tubes containing no antibiotics on the

I

t:Jl

I

N

~
~

~
~

~
~

~

....
c'
....
;::;.
~

~

0..
III

... ~ ................ 4

'<:

e.

f-l

.... +

2,;::..

day the bacilli were inoculated in Kirchner media.
(3) C indicates contamination.

W

f-l

~

c

N

....

0

~

00

(4) Numbers(f) represent colony counts on egg media.

x
I

I-'

0

I~""
I

,

....

I

I

I

fj
q-

::l

s.

~
~

Ichiro VESAKA

32

all the tubes containing agents in the same way as I had done in the control
tube and examined microscopically.
As seen in Table 6 the bacterial growth
was observed after 3 days incubation by the oil immersion system, and theantibiotic activities of streptomycin and neo-nocar din were the same.
But, ifthe dry system was used it was necessary to find out the bacterial growth for
7 days, and 0:1 this occasion streptomycin inhibited the bacterial growth at
the concentration of 1 : 2,550,000 and neo-nocardin, at 1 : 1,280,000.
The agents were dissolved in distilled water(b) Subcultivation method:and subsequent dilutions were made directly into Kirchner media.
Eachtubes were inoculated with saline suspension of tubercle bacilli.
All thetubes were incubated at 3T'C.
From the next day of incubation subcultivation onto egg media was made from all the tubes.
Colonies grown on theegg media counted.
As seen in Table 7 the antagonistic activity of streptomycin and neo-nocardin after one and three days incubation were the same,
but after five days the activity of streptomycin was a little greater than thatof neo-nocardin.
(c)

Conclusion of the studies on the in vitro activity of neo-nocardin agai-

nst tubercle bacilli:
In the in vitro screening test of antituberculous agentsagainst tubercle bacilli the determination of activity should be done as earlyas possible.
According to this principle I have devised two new methodsof testing the activity against tubercle bacilli in vitro.
By these two new
methods I have compared the antituberculous activity of neo-nocardin withthat of streptomycin.
I have found that the two antibiotics have the almost
equal activity against tubercle bacilli, it the determination of activity has
been done as early as possible.
This result indicates the new antibiotic,
neo-nocardin,

is available to the treatment of experimental tuberculosis.

Summary
1. I have found a new strain of nocardia which produced a new antibiotic
designated as neo-nocardin.
By the taxonomical studies of this strain it was
found to belong to a new species of nocardia and was designated tentatively
as Nocardia kuroishi n. sp..
Production of neo-nocar din by the surface culture of N. kuroishi A. 422
was investigated.

Antibacterial activity of the fermented broth was suppres-sed by NaCI and increased with the increase in pH of test media.
Additionof agar in the culture media increases the growth of the organism and the
production of neo-nocardin.
Glucose broth is a. good media for the production of neo-nocardin, if 0.2 or 0.3 per cent agar is added to the media.
3. Method of extraction of neo-nocardin from the fermented broth wasNeo-nocardin, a New Antibiotic:33

investigated.
After the adsorption of the antibiotic to the activated carbon
it was eluted into 4/100 N Hel methanol and then precipitated with ether.
The crude neo-nocardin hydrochloride as well as fermented broth of this organism were active against gram-positive and gram-negative bacteria.
4. In mice injected with 2 mg of crude neo-nocardin hydrochloride intraperitoneally any remarkable toxic: effects were not observed.
5. Crude neo-nocardin hydrochloride has the equal activity as streptomycin
against tubercle bacilli in vitro.
Acknowledgement
The author is indebted to Prof. Saburo UYEDA for his kind directions
throughout the work

References.
1) A. D. GARDNER &E. CHAIN

species of Proactionmyces.

Proactinomycin :a "bacteriost.atic" produced by aBrit. J. Exp. Path., 23: 123.127, 1942 cited in S. A.

WAKSMAN Microbial antagonisms and antibiotic substances.

The Commonwealth FundNew York 1945
2)

E. W. EMMART A new tuberculostatic antibiotic produced by Nocardia.
nary report.

A preli mi-

Amer. Rev. Tuberc., 55: 4, 316·333, Oct., 1947 E. W. EMMART, R. E.

KISSLING & T. H. STARK The tuberculostatic action of Nocardia extracts in whit.e
mice. J. Bact., 57: 5, 509-514, May, 1949
3) WAKSMAN & HENRIeI BERGEY's Manual of Determinat.ive Bacteriology, 6th ed., 1948