MIME-Version: 1.0 Content-Type: multipart/related; boundary="----=_NextPart_01C5CFFD.042B0190" Този документ е еднофайлова Web страница, известна също като файл за Web архив. Ако виждате това съобщение, вашият браузър или редактор не поддържа файлове за Web архиви. Изтеглете браузър, който поддържа Web архиви, например Microsoft Internet Explorer. ------=_NextPart_01C5CFFD.042B0190 Content-Location: file:///C:/236AD602/JCC0543NB.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" SCREENING THE ANTIMICROBIAL ACTIVITY OF ACTINOMYCES STRAINS, ISOLATED FROM ANTARCTICA

Jou= rnal of culture collections

Volume 4, 2004-2005, pp. 29-35

 

 

 

Screening the Antimicrobial activity of Actinomyc= etes strains isolated from Antarctica

 

Denitsa Nedialkova* and Ma= riana Naidenova

 

Natio= nal Bank for Industrial Microorganisms and Cell Cultures, 1113 Sofia, P.O.Box 239, Bulgaria;

e-mail: den37@hotmail.com<= o:p>

 

 

Summary

 

A total of 40 actinomycete strains, isolated from Antarctica, were tested for antagonistic activity against 7 Gram-positive and Gram nega= tive bacteria and yeasts, 16 phytopathogenic fungi and bacteria. During the init= ial screening 60 % of the strains showed inhibition potential against test-microorganisms. Ten of them had a broader spectrum of antibacterial activity and could be used in the development of new substances for pharmaceutical or agricultural purposes.

 <= /o:p>

Introduction

 <= /o:p>

During the last 20-30 year= s, the interest in the glacier microflora increased due to the investigation of no= vel bioactive compounds, especially antibiotics and enzymes, active in low temperatures. Because of the severe environmental conditions (low temperature, low humidi= ty, high radiation etc.) Antarctica could be used in modeling the life on other planets [1, 16, 17], investigating the intercontinental contacts = and the results of the changes between glacial and post-glacial periods as well= as their effect on the organisms.

Exploring the continental glacial samples and those of shelves and icebergs, a lot of spo= rulating and non-sporulating bacteria, some yeasts species, fungi and Actinomycetes = were found [1-5, 7, 15]. The members of the last group were not only l= ess than other microorganisms but the number of the species was also small. Mai= nly, representatives of the genera Nocar= dia, Streptomyces and Nocardiopsis <= /i>were established [2, 3, 15]. Some new Actinomyces species were isolated too, lik= e Friedmanella ctinomyce gen. nov., = sp. Nov., Friedmanella lacustris sp. Nov., Nocardiopsis aurantiacus, Modestobacterium multiseptatum gen. nov., sp.nov. [11, 12, 18, 20].

Parallel with = the investigations of the microbial diversity of the permanently frozen contine= nt, the biochemical characteristics of the microorganisms were examined too. It= was found that most of the ctinomycetes isolates possessed high proteolytic, ctinomyc and chitinase activity [8] and it was also mentioned about an antibacterial one [13]. That points the possible directions in which the lo= cal microflora researches have to be developed.

The aim of thi= s study was to investigate the antibacterial activity of ctinomycetes isolates from Antarctic soils, taken from some consecutive expeditions from 1995 to 1998.=

 

Materials and methods=

 

Microorganisms. Forty ctinomycetes strains isolated from Antarctic soils were used= in screening procedure (Table 1). For testing the antibiotic activity of the investigated strains the following test-microorganisms were used: Bacillus subtilis ATCC 6633 (NBIMCC 1709), Staphylococcus aureus NB= IMCC 3703, Pseudomonas putida NBIMCC= 1090, Ps. aeruginosa NBIMCC 3700, Micrococcus luteus NBIMCC 159, Saccharomyces cerevisiae NBIMCC 53= 7, Enterococcus faecalis NBIMCC 3915,= Escherichia coli NBIMCC 3398 [14]. Microorganisms studied in the collaboration research work with the Plant Protection Institute, Kostinbrod were also used: phytopathogenic bacteria – Clavibac= ter michiganense p.v. michiganense<= /i>, P. syringae p.v. syringae, P. syringae p.v. tabaci, Xanthomonas campestris p.v. vesicatori= ae; phytopathogenic fungi – Alter= naria sp. (cucumber isolate), Ascophy= ta melonis, Cladosporium fulvum, Cladosporium sp., Fusarium avenaceum, F. culmorum, F. moniliforme, F. oxysporum, Helmintosporium gramineum, Penicillium exspansum, Verticillium dahliae, Verticillium sp.

Media and cultivation conditions. The ctinomycetes isolates were cultiv= ated on mineral agar I [6], ISP-2 and ISP-3 [19] at 28 oC. The culture media applied for test-microorganisms were Nutrient agar I, Nutrient agar II, MRS, YPD, Potato’s agar and Saborought agar [14] and the str= ains were grown at 26 °C, 30 = °C and 37&n= bsp;°C. The dynamic batch cultivation of the actinomycetes was carried out in medium – liquid YEME in 500 ml flasks with 100 ml media at 28 °C and 240 rpm for 120 h. The fermentation medium was sown with 10 % of inoculum, taken on the 48th hour (the inoculation media had the = same composition as the fermentation one). Ethanol extracts of the mycelium were prepared.

Two variants of the agar p= late diffusion method were applied.

1. Method of agar blocks. Cylindrical pieces were c= ut out from well grown and sporulated culture of the ctinomycetes strain on solid nutrition medium. The blocks were placed on the Petri dishes deep inoculated with a fixed amount of test-microorganisms (108 cells/ml). The cultures stayed for 14 – 18 hours at 2 – 8 °C for the antibacterial substance diffusion and thereafter they were cultivated at the appropriate for the test-microorganisms temperature and duration. The antibacterial activity was measured in mm sterile zone on the 48th hour for bacteria and yeasts and on the 7th day for phytopathoge= nic fungi.

2. Well diffusion method [9]. Culture media filtrates and ethanol mycelium extracts were dropped in prepared holes of the solid nutrition medium deep inoculated with the test-microorganisms. The Petri dishes were incubated and cultivated as it w= as above described. The antibacterial activity was measured in mm sterile zone. The activity from 7 to 15 mm inhibition zone was accepted as low, 16 to 15 mm - as medium, mo= re than 25 mm = 211; as high activity.

 

Results and discussion

 

The initial antibacterial activity screening of Antarctic isolates was made aga= inst four test-microorganisms in agar medium. 40 % of the investigated strains d= id not possess any activity against the tests. Nine strains showed high inhibi= tion potential against B. subtilis  1709 – 30 and over mm sterile zone. It should be pointed out that only one of the studied strains suppres= sed the growth of E. coli 3398 and = two strains were effective against S. cerevisiae 537 but the zone diameter was smaller than 25 mm. No= ne of the Antarctic isolates exhibited activity against P. putida 1090 (Table 1).

Based on the results of the mentioned above screening 15 actinomycete strains with high inhibition potential were selected. Their activity range was checked o= ut against large number of test-microorganisms (Table 2).

 

Tab= le 1. Antibiotic activity of the actinomycete strains determined by the agar block method.

Strains

Test-microorganisms (inh= ibition zone in mm)

Strains

Test-microorganisms (inh= ibition zone in mm)

E. coli

B. subtilis

Ps. putida

Sacch. cerevisiae

E. coli

B. subtilis

Ps. putida

Sacch. cerevisiae

372= 5

22<= o:p>

42<= o:p>

0

0

372= 6

0

10<= o:p>

0

0

371= 7

0

36<= o:p>

0

0

801= 6

0

10<= o:p>

0

0

800= 7

0

34<= o:p>

0

0

371= 9

0

0

0

0

802= 2

0

32<= o:p>

0

0

372= 0

0

0

0

0

378= 9

0

31<= o:p>

0

0

372= 1

0

0

0

0

371= 8

0

30<= o:p>

0

0

372= 3

0

0

0

0

378= 4

0

30<= o:p>

0

0

372= 4

0

0

0

0

801= 3

0

30<= o:p>

0

0

378= 6

0

0

0

0

378= 7

0

30<= o:p>

0

0

800= 3

0

0

0

0

801= 0

0

30<= o:p>

0

0

800= 4

0

0

0

0

801= 8

0

29<= o:p>

0

0

800= 5

0

0

0

0

371= 5

0

28<= o:p>

0

0

800= 6

0

0

0

0

378= 3

0

28<= o:p>

0

0

800= 8

0

0

0

0

800= 9

0

22<= o:p>

0

0

801= 1

0

0

0

12<= o:p>

371= 6

0

18<= o:p>

0

0

801= 2

0

0

0

0

371= 4

0

16<= o:p>

0

0

801= 4

0

0

0

0

378= 8

0

16<= o:p>

0

0

801= 5

0

0

0

0

801= 9

0

14<= o:p>

0

0

801= 7

0

0

0

18<= o:p>

372= 2

0

12<= o:p>

0

0

802= 0

0

0

0

0

378= 5

0

11<= o:p>

0

0

3790

0

0

0

0

 

Tab= le 2. Antibiotic activity of the actinomycete strains after cultivation in fermentation media.

S= trains

Test-microorganisms (inh= ibition zone in mm)

E. coli

B. subtilis

M. luteus

Ps. aeruginosa

Ps. putida

St. aureus

E. faecalis

Sacch. cerevisiae

3= 784

0

30/301

22/22<= /p>

0

0

16/16<= /p>

15/15<= /p>

0

3= 718

0

28/30<= /p>

19/21<= /p>

0

0

10/12<= /p>

14/14<= /p>

0

8= 018

0

18/0

10/10<= /p>

0

0

0

0

0

8= 016

0

32/32<= /p>

24/24<= /p>

0

0

34/22<= /p>

16/16<= /p>

0

3= 717

0

23/24<= /p>

16/16<= /p>

0

0

0/12

12/14<= /p>

0

8= 013

0

26/25<= /p>

17/19<= /p>

0

0

10/10<= /p>

12/0

0

8= 010

0

26/30<= /p>

19/16<= /p>

0

0

10/0

12/14<= /p>

0

3= 787

0

27/30<= /p>

28/28<= /p>

0

0

20/15<= /p>

18/16<= /p>

0

8= 009

0

22/22<= /p>

15/15<= /p>

0

0

0

0

0

8= 007

0

34/26<= /p>

25/18<= /p>

0

0

18/0

16/10<= /p>

0

<= span lang=3DEN-US style=3D'mso-bidi-font-size:12.0pt'>1filtrate from liquid culture/ethanol ex= tract of the mycelium

 

For the purposes of that e= xperiment the actinonmycete isolates were cultivated in fermentation media on shaker = for 120 h. The obtained ethanol extracts of the mycelium and the filtrates from the liquid culture were used for the antibiotic activity determination= by the well diffusion method.

During the tests it was fo= und that 30 % of the strains had lost their inhibition potential perhaps due to the inconvenient liquid growth medium. Such results had been reported from other scientists too, which had found the activity reducing in comparison with th= at showed by the method of agar blocks [13]. None of the antarctic isolates suppressed S. cerevisiae 537, P. putida 1090, P. aeruginosa 3700, E. = coli 3398 but they had a significant activity against Gram-positive microorganis= ms. The highest inhibition was shown against B. subtilis 1709 and in the most of the cases both the mycelium extracts a= nd the liquid culture filtrates were active. The suppression of the M. luteus 159 was weaker as f= rom the 10 active strains only 50 % produced sterile zones larger than 20 mm and none of the= m caused more than 30 mm in diameter zones. E. coli 3398= and S. aureus  3703 were even less inhibited - on= ly 2 actinomycete isolates had higher potential against them.<= /span>

Comparing the obtained results with those of other scientists [13], it could be said = that exhibited activity against B. subti= lis 1709 varied in the same range between 18 and 34 mm, as= the last one was the highest obtained by us result.

 

Table 3. Antibiotic activity of the actinomycete strains against phytopathogenic bacteria.

Strains

Test-microorganisms (inhibition zone in mm)

P. syringae p.v. tabaci

C. michig. p.v. michiganense

X. campestris p.v. vesicatoria

P. syringae p.v. syringae

8022

0

0

0

0

3783

0

0

0

0

3784

15

16

14

18

8019

0

0

0

0

3788

0

0

0

0

8014

0

0

0

0

8018

0

10

10

13

8016

0

17

16

20

8013

0

27

26

26

8010

0

12

0

15

3789

10

12

16

20

8003

0

10

10

0

3787

14

12

0

10

8009

12

16

10

12

8007

40

35

28

36

3785

14

12

0

12

3726

12

12

14

12

3725

14

15

14

13

3718

20

20

20

18

3717

12

10

12

12

3714

10

10

12

10

3715

10

10

0

10

3719

12

12

0

0

3721

0

10

0

10

3723

0

0

0

0

<= o:p> 

Tab= le 4. Antibiotic activity of the actinomycete strains against phytopathogenic fun= gi.

Strains

Test-fungi (inhibition z= one in mm)

A. melonis

H. sporium gramineum

Cladosporium sp.

C. fulvum

P. expansum

F. avenaceum

F. moniliforme

F. oxysporum

F. culmorum

Verticillium sp.

Verticillium dahliae

Alternaria sp.

8019

0

0

0

0

0

0

0

0

0

0

0

16

3788

10

20

12

15

12

25

12

0

16

0

15

15

8014

0

12

12

15

12

30

16

0

16

0

15

14

8010

0

0

12

0

0

0

0

0

0

0

0

0

3790

0

0

0

15

0

0

0

0

0

0

0

0

3787

14

0

0

0

0

0

0

0

0

0

0

0

8008

16

0

0

12

0

0

0

0

0

0

12

0

8009

0

0

0

10

0

0

0

0

0

0

0

0

8007

25

25

18

30

20

12

15

0

20

12

20

28

3785

15

0

0

0

0

0

0

0

0

0

14

0

3718

0

10

0

0

0

0

0

0

0

0

0

0

3717

0

10

0

0

0

0

0

0

0

0

0

0

3714

0

10

0

0

0

0

0

0

0

0

0

0

3719

0

12

0

0

0

0

0

0

0

0

0

0

 

Strain 8016 possessed a higher activity against S. aureus 3703 than the described ones till that moment in literature but = in all of the cases the obtained average activities were lower.<= /p>

The antibiotic activity spectrum of the 40 strains was extended by testing their suppression potential against four phytopathogenic Gram-positive and negati= ve bacteria (Table 3). The active strains against all the tests were 25.2 = ;% of them and only two actinomycete isolates showed higher results. P. syringae p.v. tabaci was the mo= st resistant to the antibiotic treatment. The inhibition potential against C. michiganense varied between 10 = to 35 mm sterile zone an= d the most often was between 10 to 12 mm. The results were much worse than those obtained = by the other scientists [18], according which the activity ranged between 30 a= nd 40 mm in diameter. The inhibition potential shown against = X. campestris varied between 10 to 28 mm and correlated with the data publis= hed until now. In conclusion, it could be said that 20 % of the actinomycete isolates possessed antibacterial activity against Gram - positive and negat= ive bacteria, while the obtained data from other scientists were mainly about Gram-negative ones [13].

The inhibition potential of the antarctic isolates against phytopathogenic fungi was much more different from the given above (Table 4). The active actinomycetes were only 14 or 35% of the tested strains. Most of them (9 strains) inhibited one test and only three suppressed more than 9 phytopathogenic fungi. The widest activity range possessed strain 8007 and = its inhibition zones were the biggest ones - 28 to 30 mm against C. fulvum and F. culmorim. There were no actinomycetes active against F. oxysporum.

Considering the mentioned above results, it could be seen that ten from the investigated strains exhibited higher activity against pathogenic and phytopathogenic bacteria and fungi. The widest activity spectrum and the largest inhibition zones were shown by strains 8013, 3787, 3718 and 8007, and the last one possessed the best properties. Probably, the antibacterial activity of the strains is due to an antibacterial complex active against pro- and eukaryot= ic organisms. These four actinomycetes have a potential to be included in researches of new preparations with antibacterial action or for plant protection.

 

References

 

1.      = Abysov, S. S., V. I. Birya= zova, N. A. Kostrikina, 1990. Microbiology <= /i>(Moskva), 59 (6), 1091-1101 (in Russian)= .

2.      = Abysov, S. S., S. N. Filip= ova, V. D. Kuznecova, 1983. II Proc. AS USS= R, Ser. Biol., 4, 559 (in Rus= sian).

3.      = Abysov, S. S., S. N. Filip= ova, V. D. Kuznecova, 1987. Proc. AS USSR, Ser.Biol., 1, 35 (in Russi= an).

4.      = Abysov, S. S., I. N. Micke= vich, 1993. Microbiology (Moskva), 62 (6), 994-1016 (in Russian).

5.      = Abysov, S. S., I. N. Micke= vich., M. N. Poglazova, 1998. Microbiology= (Moskva), 67 (4), 547-555 (in Russian).<= o:p>

6.      = Gauze, G. 1983. Manual for determination of actinomyce= tes, Moskva: Nauka (in Russian).<= /o:p>

7.      = Bowman, J. P., A. Sharee, = M. V. McCammon, Brown, D. S. Nichols, T. A. McMeekin, 1997.Appl. Env. Microbiol., 63 (8), 3068-3078.

8.      = Chipeva, V., K. Christova,= N. Chipev, P. Moncheva, 1996. Bulgarian Antarctic Research, Life Science, 24-30.

9.      = Egorov, N., 1995. Microorganisms - antagonis= ts and biological methods for evaluation of antibiotic activity, Moskva: Vissha shkola, 200.

10.      = Kamfer, P., R. M. Koppenst= edt, 1991. J. Gen. Microbiol., 137, 1893-1902.=

11.      = Lawson, P. A., M. D. Colli= ns, P. Schumann, B. J. Tindall, P. Hirsch, M. Labrenz, 2000. Syst. Appl. Microbiol., 23 (2), 219-229.

12.      = Mevs, U., E. Stakebrandt, = P. Schumann, C. A. Gallikowski, P. Hirsch, 2000. Int. J. Syst. Evol. Microbiol., 1, 337-346.

13.      = Moncheva, P., S. Tishkov, = N. Dimitrova, V. Chipeva, S. Antonova - Nikolova, N. Bogatzevska, 2000-20= 02. J. Culture Collections, 3, 3-14.

14.      = National Bank for Industri= al Microorganisms and Cell Cultures, Catalogue. www.nbimcc.org.

15.      = Nonomura, H., 1974. J. Ferment. Technol., 52 (2), 78-98.<= /p>

16.      = Price, P. B., 2000. PNAS. Microbiology Geophysics, 97 (3), 1247-1251.

17.      = Priscu, J. C., E. E. Adams= , W. B. Lyons, M. A. Voytek, D. W. Mogk, R. L. Brown, K. P. McKey, C. D. Takacs, K.= A. Welch, C. F. Wolf, J. D. Krishtein, R. Avci, 1999. Science, 286, 2141= -2144.

18.      = Schumann, P., H. Prauser, = F. A. Rainey, E. Stakebrandt, P. Hirsh. 1997. Int. J. Syst. Bacteriology, 47 = (2), 278-283.

19.      = Shirling, B., D. Gottlieb,= 1968. Int. J. Syst. Bacteriology, 61, 313-340.

20.      = Suzuki, K., J. Sasaki, M. = Uramoto, T. Nakase, K. Komagata, 1997. Int. = J. Syst. Bacteriology, 47(2), 474-478.

------=_NextPart_01C5CFFD.042B0190 Content-Location: file:///C:/236AD602/JCC0543NB.files/header.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii"

PAGE 

 

6

 

------=_NextPart_01C5CFFD.042B0190 Content-Location: file:///C:/236AD602/JCC0543NB.files/filelist.xml Content-Transfer-Encoding: quoted-printable Content-Type: text/xml; charset="utf-8" ------=_NextPart_01C5CFFD.042B0190--