77

Novel, Efficacious Antibiotics Producedby Bacillus subtilis DB9011

Naomi NOZAKI1), 2), Sachiko SATAKE1),

Takeru IIZUKA2), and Taiji NAKAE3)

(Revised September 30, 2009, Accepted December 21, 2009)

SUMMARY : Antibiotic activities of the Bacillus subtilis DB9011 culture supernatant extracts

against 52 pathogenic microbes were screened by measuring the zone of growth inhibition of

the pathogens under a sterile paper disc loaded with the supernatant extract. The first

scientific evidence for the effectiveness of the DB9011 culture extract against Porphyromonas

gingivalis, Clostridium difficile, Propionibacterium acnes, and Legionella pneumophila is

presented here. These organisms are causative pathogens of periodontal disease, Clostridium

difficile-associated colitis, acne, and pneumonia, respectively. The findings from the present

study will contribute to the development of more efficacious and safer pharmacotherapeutic

agents for these diseases.

Key words : Bacillus subtilis, Antibiotics, Porphyromonas gingivalis, Antibiotic activity

screening

1) School of Health Sciences, Gunma University, 3-39-22 Showa-machi, Maebashi 371-8511, Japan: 2) AHC Co.,Ltd,343-

1 Koaigi-machi, Maebashi 371-0831, Japan: and 3) Kitasato Institute University, S105, 1-15-1 Kitasato, Sagamihara

228-8555, Japan

Ann Gunma Health Sci 30：77－81，2009

INTRODUCTION

Since the advent of the antibiotic, bacitracin, in

19431) different strains of Bacillus subtilis (B.

subtilis) have provided a good source for a variety of

antibiotics and other pharmaceuticals.2-6)

Furthermore, B. subtilis culture itself has been used

widely for dietary supplements for human, livestock,

and poultry.7-10) In fact the widely consumed,

centuries old Japanese delicatessen/health food,

Natto, is composed of odoriferous, sticky fermented

soybeans prepared by means of one of the many B.

subtilis strains, commonly known as Bacillus natto.

Historically purported human health benefits of Natto

include growth promotion and curing of intestinal

diseases, gingivitis, heart diseases and a few more.

Not long after the Typhoid Mary incident in the

U.S., Japanese investigators reported that feeding the

Typhoid patients Natto eradicated completely the

causative pathogen, Salmonella Paratyphi B, from the

human intestinal tracts.11),12) Scientific reports in

support of some of the purported human health

benefits are appearing in both Japanese and Western

scientific journals in recent years.13-17) Furthermore,

the efficacy and safety of B. subtilis cultures and

subtherapeutic doses of antibiotics from the culture

as a growth promoter have been reported.3),18),19) In

Japan, the safety of B. subtilis culture for human

consumption was recognized based on the long

experience with Natto. In the U.S., on the other hand,

the U.S. Environment Protection Agency (EPA)

assessment defined that B. subtilis strains as a group

are benign organisms as they do not possess traits

that cause disease.20)

B. subtilis DB9011 was isolated from Japanese soil

in 1990,4) and provided a good source for the

production of antibiotics and other

78

pharmaceuticals.3),4) However, the full potential for

production of novel antibiotics has not been explored.

This report describes a thorough assessment of a

broad range of antibiotic spectrum found in the

DB9011 culture supernatant, giving particular

attention to the historically purported human health

benefits of B. subtilis and products derived from it.

MATERIALS AND METHODS

DB9011 cells were grown on Mueller-Hinton agar

plate (MHA, Eiken Chemicals, Tokyo), and a single

colony was inoculated into Mueller-Hinton broth

(MHB, Difco, USA). The flask was incubated

aerobically at 35℃ on a rotator shaker at 200 rpm for

desired time periods, which were dependent upon the

purpose of each preparation. The cells were removed

by centrifugation at 15,000 rpm for 15 min, and the

supernatant was filtered through a 0.45 μm filter

(Advantec. Tokyo).

The filtered DB9011 supernatant was mixed with 1

N HCl until the pH reached 3.0. The precipitated

material was separated by centrifugation for 10 min at

15,000 rpm. The active component was extracted by

solubilizing the pellet in an aliquot of 80% ethanol to

obtain 300-fold concentrated supernatant solution (300

x extract).

The following preliminary experiments were

performed in order to establish the rationale for

choosing DB9011 and the rest of the experimental

protocols. Porphyromonas gingivalis JCM 8525 (P.

gingivalis)---the strain that had been shown as the

main causative agent of periodontal diseases 21)---was

used as the test strain for the determination of

optimum condition for the antibiotic production by

DB9011. In this series of experiments 20 μl of the

filtered DB9011 supernatant were used for the

determination of zone of growth inhibition (ZOGI)22-24).

The time course of anti-P. gingivalis activity during

the DB9011 growth period showed that the activity

peaked during the stationary phase at the 24th hour

after the inoculation, and thereafter the activity

declined gradually during the 48 h growth period.

Table 1：Summary of test strain culture conditions

79

Therefore the antibiotic activities of 4 other

commonly available B. subtilis strains that consisted

of ATCC6633, BN1001, KUBOTA, and OUV23481

were compared with that of DB9011. Under the

conditions of the experiments the ZOGI of DB9011

was 25 mm in diameter whereas that of ATCC6633

was 10 mm and others were equal to or less than 8

mm.

The antibiotic activities of the 300 x extract of

DB9011 against 52 test strains of potential pathogens

were assayed using specific agar medium and growth

conditions appropriate for each test strain.25,26) The

conditions of each test strain culture are codified and

summarized in Table 1.

RESULTS AND DISCUSSION

Table 2 shows the antibiotic activities assay results.

Anaerobic organisms seemed to be more frequently

susceptible than those in other groups. Common

nosocomial pathogens in the aerobic and facultative

anaerobic group seemed less susceptible to the active

component of the 300 x extract than those in the

anaerobic group. Furthermore, it is interesting to note

that the potent antibiotic activity of B. subtilis

DB9011 extract against P. gingivalis shown in this

study is - consistent with the purported effectiveness

of Natto, the Japanese delicatessen referred,7-12)

earlier. Our findings also corroborate the pre-

antibiotic era Japanese investigators’ reports 11),12)

concerning the effectiveness of a Natto diet for

eradicating intestinal Samonella Paratyphi B.

Among the organisms most sensitive to the 300 x

extract were P. gingivalis, C. difficile, L.

pneumophila, and P. acnes. Currently, there are no

satisfactory pharmacotheraputic regimens to treat

the periodontal disease caused by P. gingivalis.21)

Clostridium difficile-associated colitis can be lethal,

and the drugs of choice are vancomycin and

metronidazole. These antibiotics are produced from

Streptomyces orientalis (Amylcolaptosis orientalis)

and by the organic chemical synthesis method,

respectively. This observation suggested that the

Table 2：Suscetibility of pathogens to 300 x extract of DB9011 culture supernatant

80

anti-P. gingivalis component in the DB9011 culture

extract could be a novel vancomycin-like antibiotic.

The search for new, more efficacious and safer

antibiotics is always needed in preparation for the

emergence of antibiotic resistant strains.

The recent reports concerning the mechanisms,

efficacy, and safety of B. subtilis culture and the

products derived from the culture for human health

benefits cited here13-19) together with the findings in

this report suggest that the anti-P. gingivalis, -C.

difficile; -L. pneumonia, and -P. acnes substances

described here are likely novel, efficacious, and safer

pharmacotherapeutic agents.

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