Novel, Efficacious Antibiotics Produced
by Bacillus subtilis DB
9011
Naomi NOZAKI
1), 2), Sachiko SATAKE
1),
Takeru IIZUKA
2), and Taiji NAKAE
3)(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 pneumophilais 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 37Co.,Ltd,343-1-083Co.,Ltd,343-1, Japan: and 3)Kitasato Institute University, S105, 1-15-1 Kitasato, Sagamihara
228-8555, Japan
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. subtilisDB9011 was isolated from Japanese soil in 1990,4) and provided a good source for the production of antibiotics and other
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.
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-associatedcolitis 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
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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