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Detection of antitumor and antimicrobial activities in marine organism associated actinomycetes isolated from the Taiwan Strait, China

Zhonghui Zheng, Wei Zeng, Yaojian Huang, Zhiyuan Yang, Jun Li, Huirong Cai, Wenjin Su
DOI: http://dx.doi.org/10.1111/j.1574-6968.2000.tb09173.x 87-91 First published online: 1 July 2000

Abstract

The purpose of this work was to screen the actinomycetes having antitumor or antimicrobial activity, which were isolated from the surface, epidermis and intestines of sea plants and animals collected from the Taiwan Strait, China. Antitumor activity was studied by the MTT assay and DNA target activity was studied by the biochemical induction assay while antimicrobial activity was determined by observing bacterial and fungal growth inhibition. 20.6% of marine actinomycete cultures displayed cytotoxic activity on P388 cells at dilutions at and below 1:320 and 18.6% on KB cells. 2.96% of marine actinomycete cultures displayed inducing activity. Among all marine actinomycetes isolated, the genus Micromonospora has the highest positive rate of inducing activity. However, most antimicrobial activity was found in the genus Streptomyces. These results indicate that marine organism associated actinomycetes could be a promising source for antitumor and antimicrobial bioactive agents.

Keywords
  • Marine actinomycete
  • Antitumor
  • Antimicrobial
  • Taiwan Strait

1 Introduction

As a great promising source for new natural products which have not been observed from terrestrial microorganisms, marine bacteria are being developed for the discovery of bioactive substances with new types of structure, with growing intensive interest. The achievements have been well reviewed [1,2], where many new antibiotic were obtained from actinomycetes. Proven by numerous isolates from soil, actinomycetes merit competitive biosynthetic capabilities so that marine actinomycetes naturally become the prime candidate in screening as producers of new natural products. Meanwhile, there are also documents demonstrating that many marine natural products previously ascribed to other organisms are secondary metabolites of bacteria associated with the organisms [3,4]. Thus, the actinomycetes associated with marine plants and animals are expected to be a potential source for new natural bioactive agents. However, few studies in this area have been conducted, the majority of previous studies focused on sea sediment derived actinomycetes. So, further systematic investigation of symbiotic marine actinomycetes is necessary, because it will not only provide us with much useful ecological information, but would also be a way of finding new natural products with a higher hit rate. Herein we present the data of our screening for marine actinomycetes having antimicrobial and antitumor activity isolated from sea plants and animals collected from the Taiwan Strait, China.

2 Materials and methods

2.1 Collection of samples and isolation of strains

Except the sharp, Mustelus manazo, purchased from a local market, all other marine plant and animal samples, including sea hare, Aplysia dactylomela, sea anemone, Actiniaria, and sea plants, Ulva lactuca, enteromorpha and Gracilaria verrucosa, were collected at three sampling sites in the intertidal zone of Xiamen Island, which is located on the southwest coast of the Taiwan Strait, China.

In order to avoid replication of identical strains in our investigation, the following collection and isolation procedure was adopted. Five sampling points (30×30 m2 each) were set in every sampling site. A total of 100–200 g plant or animal samples was randomly collected in each sampling point. Samples with equal weight of the same species but from different sites, after washing with sterilized seawater to eliminate impurities such as sand, were mixed and divided into five parallel portions on average.

To isolate the actinomycetes from the surface, epidermis of sea hare, sea anemone, and the other sea plants, all parallel portions of samples were ground into a paste. After centrifugation. the supernatant of the paste was diluted (102–104) and plated on solid Emerson's medium (five plates to each dilution). Plates were incubated at 28°C for 21 days. However, the method used to isolate the actinomycetes in intestines of sharp was modified from that of NoGuchi [5] while the selective medium was Emerson's agar medium as well.

2.2 Identification of actinomycetes

Strains were preliminarily identified according to traditional morphological criteria, including characteristics of colonies on the plate, morphology of substrate and aerial hyphae, morphology of spores, pigment produced and so on, while the wall chemotype and whole cell sugar pattern of the active strains were further examined [6].

2.3 Fermentation and treatment of the fermentation broth

The fermentation medium contained soluble starch 20 g, soybean powder 15 g, glucose 5 g, yeast extract 2.5 g, CaCO3 1 g, seawater to 1 l, pH 7.5. The strains were cultured in the fermentation medium for 5 days at 28°C. Crude culture broth was treated with ultrasound and centrifuged at 10 000×g for 30 min. The supernatants were passed through a filtration membrane (d=0.22μm, Minipore) before their bioactive activity was assayed.

2.4 Detection of cytotoxic activity and DNA target activity

The cytotoxic activity of fermentation broths was studied by the MTT assay. Drugs were kindly donated by the Cancer Research Center of Xiamen University. The cell lines used were murine leukemia P388 cells and three human cancer cell lines, KB cells, HLF cells and CNE cells. The MTT assay protocol was adapted from that described by Mosmann [7]. The optical density (OD) of the wells was measured with a microplate reader (M-3550, Bio-Rad) at 595 nm with 655 nm as reference. The growth inhibition rate was calculated by the following equation: Embedded Image IC50 or ID50 was defined as the drug concentration or the fermentation broth dilution that resulted in at least 50% growth inhibition.

The DNA target activity of fermentation broths was studied by biochemical induction assay (BIA) and the BIA protocol was adapted from that of Elespuru and White [8]. Escherichia coli strain BR513((λplacZ cl+ PRt11) pro-lacΔuvrBΔenvA azi thi rpsL gal) was obtained from ATCC and cultured in LBE medium.

Before the MTT assay was used to test the antitumor activity of the fermentation broth, its reliability and sensitivity were verified by seven well-known antitumor agents, bleomycin, cisplatin, mitomycin C, methotrexate, 5-fluorouracil, cytarabine hydrochloride and vincristine sulfate, and five antibiotics, chloramphenicol, gentamicin, lincomycin, micronomicin and kanamycin, and three organic solvents without antitumor activity, dimethyl sulfoxide (DMSO), methanol and ethanol.

In the BIA spot test, three agents definitely known to interact with DNA, bleomycin, cisplatin and mitomycin C, were chosen as positive controls while the negative controls were inhibitors of protein synthesis or other drugs having indirect effects on DNA, including methotrexate, 5-fluorouracil, cytarabine hydrochloride, vincristine sulfate and cyclophosphamide.

2.5 Determination of antimicrobial activity

Against a battery of five bacteria, two yeasts and one filamentous fungus, antimicrobial activities of the isolated marine actinomycetes were preliminarily determined by agar streaking, then identified using the double-layer method [9,10]. The test bacteria included Bacillus subtilis ATCC 9372 (BS), Bacillus licheniformis CMCC(B) 63519 (BL), Staphylococcus aureus ATCC 25923 (SA), E. coli ATCC 25922 (EC), and Vibrio anguillarum XM 9024 (VA). The fungi tested were Saccharomyces cerevisiae 9763 (SC), Candida albicans As 2.538 (CA) and Aspergillus niger ACCC 30005 (AN).

3 Results and discussion

3.1 Distribution of actinomycetes

In all, 439 actinomycetes were isolated in our survey. Total counts of actinomycetes recovered from different samples ranged from a maximum of 5.4×105 cfu g−1 sample (surface of the sea hare, Aplysia dactylomela) to a minimum of 5.0×102 cfu g−1 sample (the marine plant, Gracilaria verrucosa). Strains belonging to the genera Streptomyces and Micromonospora, especially the former, represented the majority. Out of the total, only 43 (9.8%) other strains, not belonging to these two genera, was isolated.

3.2 Screening of marine actinomycetes having cytotoxic activity by the MTT assay

For all the standard antitumor agents, the IC50 value was less than 25 μg ml−1 against at least one cell line and P388 cells were the most sensitive to the antitumor agents among the cell lines tested (Table 1). The sensitivity of the four cell lines to five antibiotics without antitumor activity and three organic solvents is summarized in Table 2. Only the IC50 value of chloramphenicol was less than 25 μg ml−1 against the KB and HLF cell lines, other antibiotics and organic solvents had no effects on the cancer cells tested. These data indicate that the MTT assay utilized in our study was suitable for screening antitumor agents with cytotoxic activity.

View this table:
Table 1

In vitro antitumor activity of marine actinomycetes determined by the MTT assay

Compound or strainIC50 (μg ml−1)/ID50a
P388 cellsKB cellsHLF cellsCNE cells
Bleomycin1.000.40<0.0050.65
Cisplatin0.401.251.000.70
Mitomycin C0.0050.150.400.17
Methotrexate<0.0050.030.040.12
5-Fluorouracil<0.1258.000.12525.00
Cytarabine hydrochloride2.0020.0010.0050.00
Vincristine sulfate<0.00250.0110.002.00
MA11641:20 4801:20 4801:20 4801:8 200
MA4291:20 4801:20 4801:20 4801:4 900
MA3501:20 4801:20 4801:7 9001:2 200
MA10231:3 8001:8001:2 2001:400
  • aIC50 for control drugs; ID50 for fermentation broths.

View this table:
Table 2

Effect of non-antitumor agents on tumor cells tested by the MTT assay

Compound (25 μg ml−1)Growth inhibition rate (%)
P388 cellsKB cellsHLF cellsCNE cells
Chloramphenicol37.592.972.338.0
Gentamicin12.69.210.16.7
Lincomycin20.003.75.4
Micronomicin21.010.811.29.4
Kanamycin16.09.713.18.8
DMSO5.52.66.424.9
Methanol003.92.0
Ethanol2.57.213.60

Then, fermentation broths of 360 marine actinomycetes, randomly selected from all 439 isolates, were tested for cytotoxicity. Table 3 shows the number and percentage of the broths at a dilution of 1:320 displaying activity as a function of the growth inhibition rate. 20.6% of marine actinomycete cultures displayed cytotoxic activity on P388 cells at dilutions at and below 1:320 and 18.6% on KB cells. Four broths from the cytotoxic-positive strains were chosen to study their effect on the growth of four cancer cell lines and the ID50 dilutions for these broths against cancer cells ranged from 1:800 to greater than 1:20 480 (Table 1). P388 cells were the most sensitive among the cell lines. The ID50 dilutions for these broths against P388 cells ranged from 1:3800 to greater than 1:20 480.

View this table:
Table 3

Screening of fermentation broths (diluted to 1:320) with cytotoxic activity from marine actinomycetes by MTT assay

Number of strains with cytotoxic activity (%)
P388 cellsKB cells
Number of strains tested360 (100)360 (100)
Growth inhibition rate=100%29 (8.1)12 (3.3)
100%≥growth inhibition rate≥90%10 (2.8)6 (1.7)
90%≥growth inhibition rate≥50%35 (9.7)49 (13.6)
Total74 (20.6)67 (18.6)

3.3 Screening of marine actinomycetes having DNA target activity by BIA

The BIA spot test is considered an in vitro mechanism-based screening method with simplicity, sensitivity, rapidity and reproducibility to test selectively DNA target antitumor activity of fermentation broths [8]. Table 4 demonstrates that the BIA test was selectively sensitive to those agents that interact with DNA.

View this table:
Table 4

Phage induction of antitumor agents tested by BIA

CompoundSolventAmount applied (μg per spot)Minimal inducing amount (μg per spot)DNA interaction
BleomycinWater0.01–100.01+
Cisplatin0.15 M NaCl0.4–502+
Mitomycin CMethanol0.001–10.01+
MethotrexateWater0.1–10>10
5-FluorouracilDMSO0.025–25>25
Cytarabine hydrochlorideWater0.25–250>250
Vincristine sulfateWater0.1–5>5
CyclophosphamideDMSO0.4–400>400

All 439 fermentation broths of marine actinomycetes were subjected to screening to detect their potential DNA target activity using the BIA spot test and 2.96% of marine actinomycete cultures displayed inducing activity (Table 5). The selectivity of BIA resulted in a lower positive rate of screening compared with the MTT assay. It is noticed that in our study the genus Micromonospora has a higher positive rate of inducing reaction than the genus Streptomyces.

View this table:
Table 5

Screening of fermentation broths with inducing activity from marine actinomycetes by BIA

Number of strains testedNumber of strains with inducing activity
Micromonospora685 (7.35%)
Streptomyces3288 (2.44%)
Other genera43none
Total43913 (2.96%)

3.4 Antimicrobial activities of the isolated marine actinomycetes

Only 39 marine actinomycetes were randomly picked out from the total of hundreds of isolates to test antimicrobial activity, because we are more interested in antitumor antibiotics. Out of these tested strains, 17 strains (43.6%) having antimicrobial activity were observed. They belong to Streptomyces and Micromonospora, the former being the majority (Table 6). The Streptomyces isolates can be classified into five groups, flavus (63.6%), albosporus (11.7%), roseosporus, viridis and hygroscopicus. Among all 17 antimicrobial isolates, 11 (65%) exhibit inhibition activity against Gram-positive bacteria and eight (47%) against Gram-negative bacteria, while four strains (23.5%) exhibit antimicrobial activity against both Gram-positive and Gram-negative bacteria. Antifungal activity was present in nine isolates (52.9%), while two of them inhibited the growth of two fungal strains. Streptomyces and Micromonospora, especially the former, represented the major genera identified.

View this table:
Table 6

Identification and antimicrobial spectra of marine actinomycetes

Strain No.GenusTested strainsa
BSBLSAECVASCCAAN
A3Streptomyces sp.++++
A4Streptomyces sp.++
A7Streptomyces sp.+
A9Streptomyces sp.++++++++
A10Streptomyces sp.+++++++
A12Streptomyces sp.+
A15Streptomyces sp.+
A16Streptomyces sp.+++++
A17Streptomyces sp.+
A18Streptomyces sp.+++++
A20Streptomyces sp.++
A24Streptomyces sp.++
A26Streptomyces sp.+++
A29Streptomyces sp.++
A32Streptomyces sp.+++
A6Micromonospora sp.+++
A30Micromonospora sp.++++
+, inhibition zone≤1.0 cm; ++, 1.0 cm<inhibition zone≤2.0 cm; +++, inhibition zone≥2.0 cm.
  • aFor the full names see Section 2.5.

By the late 1990s, there had been about 40 new bioactive microbial products from marine organisms reported [1,2], near half of them were found in actinomycetes, demonstrating that the hit rate of new antibiotics from marine actinomycetes is higher than from other microbial species. Something similar had occurred with studies on terrestrial microorganisms. However, the majority of these marine actinomycetes were isolated from sea sediment.

On the other hand, more than a third of all discovered new bioactive microbial products from the sea were derived from the bacteria associated with marine animals, especially invertebrate animals, or marine plants. These symbiotic or commensal bacteria, in many instances, constitute the normal flora associated with the host and chemically defend their microhabitat while protecting their host from pathogenic microorganisms by producing secondary metabolite compounds. A typical example is isatin, produced by a symbiotic marine bacterium identified as Alteromonas sp., which protects embryos of the shrimp Palaemon macrodactylus from infection by the pathogenic fungus Lagenidium callinectis[11]. Moreover, some antibiotics previously ascribed to marine organisms, such as sponges, the proliferative producer of the natural product, were finally traced to its symbiotic bacteria [3,4]. Considering these cases, it could be said that the marine organism associated actinomycetes are a promising source for antimicrobial and antitumor bioactive agents. More attention should be paid to these ecological species, though further scientific evidence needs to be produced to verify the symbiotic or commensal relationship between these actinomycetes and their hosts. Several bioactive agents have been discovered from these actinomycetes, from soft coral [12,13], jellyfish [14], and sponges [15]. Ecological surveys were conducted as well, one of them was the investigation of the diversity of actinomycetes on seaweeds [16].

In our study, approximately 43.6%, 20% and 3% of tested marine actinomycete cultures displayed antimicrobial activity, cytotoxic activity and inducing activity, respectively. The positive rate of antimicrobial activity is higher than the data from surveys of sediment derived actinomycetes, around 16% in Pisano et al.'s study [9] and 18% in that of Ellaiah et al. [17]. Among the strains tested, strain MA350 was noted for its high cytotoxic activity (the ID50 dilutions against P388 cells and KB cells were greater than 1:20 480, Table 1) and high inducing activity (inducing activity was similar to that of 1 mg ml−1 of cisplatin, data not shown), suggesting that the cytotoxic effects may result from its interaction with DNA. The characteristics of antitumor bioactive agents produced by MA350 will be studied.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant 39670015), the Fujian Province Natural Science Foundation of China (Grant F99032) and the Research Fund for the Doctoral Program of Higher Education, China.

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