Production of antibacterial activities by two ...

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Production of antibacterial activities by two Bacillariophyceae grown in dialysis culture Institut National de la recherche scientijique, I.N.R.S.-Sante', Universitt du Qukbec, Montre'al, Que'., Canada H1N 3M5 Insti tut National de la recherche scientijique, I .N .R.S. -Oce'anologie, Universite' du Que'bec, Rimouski, Que'., Canada G5L 3A1 AND

Institut Armand-Frappier, Centre de recherche en bacte'riologie, Universite' du Que'bec, Laval, Quk., Canada H7V 1B7 Accepted November 22, 1982 COOPER, S., A. BATTAT, P. MARSOT, and M. SYLVESTRE. 1983. Production of antibacterial activities by two Bacillariophyceae grown in dialysis culture. Can. 3. Microbiol. 29: 338-341. Using a dialysis culture system for marine algae which allows for the recovery of high cell yield, we detected in the aqueous phase of the algal extracts an active component which inhibited gram-negative bacteria. Results show a direct relation between cell growth phase at time of harvest and the activity observed. It was possible to confirm previous results whereby BaciZlariophyceae produce lipophilic substances inhibiting gram-positive terrestrial and marine bacteria. These findings corroborate the observation that Skeletonema costatum is more active than Phaeodactylum tricornutum against Staphyloccoci. COOPER, S., A. BATTAT, P. MARSOT et M. SYLVESTRE. 1983. Production of antibacterial activities by two BacilZariophyceae grown in dialysis culture. Can. 3. Microbiol. 29: 338-341. Nous avons rnis en dvidence diverses activitbs antibiotiques chez deux especes de phytoplancton marin cultivC en dialyse. Le rendement devC du syst&mede culture A dialyse utilisk a pennis de dCceler dans les extraits aqueux, un composant inhibiteur envers les bact6ries gram-negatives et d'Ctablir une relation entre la phase de croissance de I'algue et la production des activitds antibact6riennes. De plus, nos rdsultats viennent appuyer les observations pkddentes ii l'effet que les Bacillariophycdes eroduisent des substances lipophiliques inhibant la croissance de bactCries terrestres gram positives et de batteries marines. Egalernent, enversles staphylocoques,Skeletonema costatum s'est avCrCe plus efficace que l'algue Phaeodactylum tricornuturn.

(1972), all other investigators mentioned previously Introduction It was previously shown that several planktonic were using algal cultures grown in batch culture. Recently, Marsot et al. (1981a, 1981b) have marine algae can produce cell-associated antibacterial developed a simple adaptation of dialysis culture for the substances. Hence, in mixed cultures, Skeletonemu costatum (Grev.) inhibited the growth of several marine production of algal cells. This technique allows for the bacterial strains, including Pseudornonas and Vibriu recovery of high cell yield derived from cultures grown (Kogure et ale 1979). Gauthier et al. (1978) have shown on natural seawater. This technique was used to the presence of lipophilic antibiotics associated with investigate the possibility of antibacterial activities in Chaetoceros lauderii (Ralfs) cells, and Duff et al. these cultures. (1966) have also demonstrated that several marine algae Material and methods belonging to different algal classes produced cellThe algal strains Phaeodactylum tricornutum and Skeletoassociated antibacterial activities. Caccamese and Azzolina (1979) also made a similar observation. In nema costatum used in this work were obtained from the Bedford Institute of Oceanography, Dartmouth, N.S. Strains all cases terrestrial gram-positive and marine bacteria were maintained in the F/2 medium described by Guillard and were the most sensitive test organisms. Apart from Ryther (1962). cell-associated antibacterial activities, Berland et ul. The dialysis cultures were grown aseptically under the (1972) were the first to report the isolation of free conditions previously described by Marsot et al. (198 1a). antibacterial substances from the culture filtrate of Running seawater (containing 5.0 to 12.0 mM of N-NO3 and Stichochrysis immobilis (Chrysophyta) grown in fer- 0.5 to 2.0 mM of P-PO4, with salinity values ranging from 25 mentors. Except for the above work of Berland et al. to 29 %o) was circulated through the dialysis units (hollow fiber ' ~ u t h o rto whom correspondence should be addressed.

hemodialyzer manufactured by Hospal Med. Corp., Littleton, CO) at a flow rate of 10 L/h to supply fresh nutrients. The

COOPER ET AL.


culture broth inside the growth chamber was circulated between the fibers of the dialysis units at a flow rate of 6 L/h. The cultures were thermostatically controlled at 12 k 40.5"C and exposed to natural light ranging from 3000 to 80 000 lx. The growth chamber was inoculated with 40 to 60 mL of a log phase algal culture grown in F/2 medium. The 15-L growth chamber was connected to three dialysis units (total exchange area = 3.3 m2). The cells from 15 L of cultures were harvested by centrifugation at one of the following stages: early log phase, late log phase, or stationary phase. The culture media was separated from the cells by centrifugation at 12 000 x g and then extracted with a mixture of chloroform-methanol (2: 1, v/v). These extracts as well as similar control extracts from 12 L of seawater were tested for activity. The cells were washed with a citric acid (0.1 M ) - disodium phosphate (0.2 M ) buffer at pH 7.0 and then suspended in 100% ethanol and stored at -20°C in the dark. At the time of extraction, the cells were concentrated by centrifugation and the ethanolic supernatant was evaporated. The residue together with the cells was extracted with chloroform, methanol, and water by the method described by Bligh and Dyer (1959). The samples were extracted with a monophasic solution of chloroform-methanol-water (1:2:0.8, v/v). The cells were removed by centrifugation and to the supernatant was added chloroform and water (1:1, v/v) yielding a biphasic system of chloroform-methanol-water (2:2: 1.8, v/v). The aqueous and the lipophilic phases were separated and evaporated to dryness. The lipophilic phase was dissolved in a minimal volume of 1 to 5 mL of chloroform-methanol (2: 1, v/v) and the aqueous phase in a minimal volume of 2 to 3 mL of water depending on the quantity of cells extracted. Both the aqueous and organic phases of the cell extracts were held at -20°C in the dark until required for analysis. They were then tested against dzferent bacterial species using the agar-disc diffusion method. The terrestrial bacterial strains used as test organisms were from the bacterial research center (Institut Armand-Frappier, Laval, Que.) culture collection and included Pseudomonas aeruginosa , Escherichia coli ATCC 9980, Bacillus subtilis, and Staphylococcus aureus ATCC 6340. The marine bacteria tested were Alteromonas communis, Alteromonas haloplanktis, Vibrio parahaemolyticus, Vibrio jscheri, Pseudomonas marina, and Alculigenes cupidus. These cultures were graciously provided by the Microbiology Dept., Macdonald Campus, McGill University. A Mycoplasma oraleae strain from our own collection was also included among the test organisms. Nutrient agar plates (IAF-Production, Laval, Que) were used for the cultivation and propagation of all terrestrial bacterial strains. Mycoplasma oraleae was grown on Difco PPLO agar (Detroit, MI) supplemented with 0.1% sodium acetate, 0.5% Difco yeast extract, and 5% horse serum (IAF-Production). Difco marine agar was used for the growth of all marine strains except Alteromonas haloplanktis which was cultivated on the medium described by Gow et al. (1973). Antibiotic tests using terrestrial strains were incubated at 37°C; those using marine bacteria were incubated at 29OC. Mycoplasma oraleae was grown anaerobically under an a&osphere of nitrogen containing 15% (v/v) C02. Bacillus subtilis spores were prepared by the method of Gould (1969).

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339

Sterile paper discs (7 mm diameter) were first dipped into the solution to be tested. In the case of water solution, they were immediately deposited onto the surface of seeded agar plates inoculated with cells in the log phase of growth. Depending on the test organism involved the plates were seeded with or lo8 cells. When testing the lipophilic extract solubilized in organic solvent, the discs were first evaporated in vacuo for 6 h before they were deposited onto seeded agar plates containing 1o6or 1o8cells as above. In both cases, the plates were incubated 18 to 24 h at temperatures of 37°C or 29°C depending on the test organisms before reading the inhibition zone diameter.

Results and discussion The spectrum of activity of algal cell extracts (aqueous and organic phases) from Pheodactylum tricornuturn and Skeletonema costaturn sampled at different growth phases are shown in Table 1. In this table, the zone diameter is given in millilitres and it is the mean of duplicate results. It must be emphasized, however, that the zone diameters listed in this table do not reflect a quantitative measurement of the antibacterial activities per unit weight of the cell mass extracted. Rather, they reflect the activities of cell extracts from 15-L cultures regardless of the cell mass at the time of harvest. For instance, in the case of P. tricornuturn the dry weight of cells harvested from 15 L of culture was 0.04 g at the early log phase and 0.46 g at the late log phase, but in both cases, after extraction of the cells, the final volume of the organic and aqueous phases was adjusted to 1I11L and 2mL, respectively. However, it is interesting to note from Table 1 that the aqueous phase of the P. tricornuturn extracts was as active in the early log phase as in the late log phase. Then, during the stationary phase, there was no activity against marine bacteria and a trace of activity against E. coli. The aqueous phases of P. tricornuturn cell extracts was thus more active against marine bacteria in the log phase of growth than in the stationary phase while the reverse relationship was observed in the case of S. castatum cell extracts. In addition, unlike the aqueous phases of P. tricornuturn cell extracts, the aqueous phase of S. costaturn cell extracts showed a broader spectrum of activity, being active against P. aeruginasa, M. oraleae, and gram-positive bacteria. It thus appears that not only is the antibacterial activity of the aqueous phase of the cell extracts specific for the algd species tested, but the spectrum of activity of these preparations is also related to the time of harvest of the culture. Debro and Ward (19791, among the few authors to have studied the effect of the growth phase of algae on the antimicrobial activity of the cell extracts, reported similar findings for cultures of freshwater green algae. For both algd strains that we tested the lipophilic phase was mostly active in the stationary phase of

340

CAN. J. MICROBIOL. VOL. 29, 1983

'

-

TABLE1. Antimicrobial spectrum of activity of algal cell extracts*

-

Pheodactylum tricornutum

Organic phase


Aqueous phase

Bacillus subtilis (spores) Staphylococcus aureus Escherichia col i Pseudomonas aeruginosa Mycoplasma oralae Alteromonas communis Vibrio parahaemolyticus Vibrio Jischeri Pseudomonas marina Alcaligenes cupidus Alteromonas haloplanktis

1

2

3

-

-

-

-

-

-

-

-

7.5

-

-

-

-

-

-

10

10

-

Skeletonema costatum

Aqueous phase

1

2

3

1

-

7.5

7.5

-

7.5

9

-

-

-

-

-

7.5

-

-

7.5

-

-

7.5

-

-

7.5

-

2

7

Organic phase 3

1

8

16

N.D. N.D. N.D.

3

2

8

7

N.D. N.D. N.D. -

-

--

*As determined by the agar diffusion assay using 7-mm paper discs. Bheodactylum tricornuturn and S. costarum algal cells from early log phase (1). late log phase (2), or stationary phase (3) were extracted by the methods of Bligh and Dyer (1959) as described in the text and the organic and aqueous phases were tested for activity. ?The numbers refer to the diameter of the inhibition zone in millimetres also including the diameter of the disc. The minus signs indicate no activity; N.D., not determined.

growth. Although there were some slight differences in the spectra of activity, these lipophilic preparations were active solely against gram-positive and marine bacteria. The only important difference in the spectrum of activity of lipophilic preparations obtained from both organisms was the inhibitory activity against M. oralae and S . aureus. Except for the activity against Mycoplasma which does not appear to have been reported previously, the organic phases of the cell extracts of both algal species reported here showed an antimicrobial spectrum of activity resembling that reported for other algal species grown by other culture methods (Gauthier et al. 1978; Gauthier 1980).Moreover, except for differences related to the time of harvest and variation in the extraction procedures, our- data corroborate those obtained by Duff et al. (1966) with P . tricornutum and S . costatum. Their study showed that both algae were predominantly active against marine bacteria, while S . costaturn was more active against S . aureus than was P . tricornutum. Our data differ from those of Duff et al. (1966) in only two points. First we detected an antistaphylococcal activity in the aqueous phase of S .

costaturn in addition to the corresponding activity in the lipophilic fraction. However, the different extraction procedures used are probably the cause of this discrepancy. The second divergent observation pertains to the activity we noted against gram-negative terrestrial bacteria in both S . costatum and P . triconzuturn aqueous phases. These activities were, however, only found in the late stationary phases of growth which were not analyzed by Duff et al. (1966). With respect to the free active components produced by algal cells as was found in the culture medium of Stichochrysis immobilis (Berland et al. 1972), our culture technique did not allow the recovery of active components with molecular weights smaller than 10000 since these would pass through the dialysis membrane used. In our study, the supernatant from the 15-L cultures was extracted repeatedly with (2: 1, v/v) chloroform-methanol mixture, evaporated, solubilized in a minimal amount of the same solvent system, and tested for activity against the same bacterial strain as the cell extracts. In these preparations, few inhibitory zones were detected against marine bacteria and B. subtilis

COOPER ET AL.


spores. In two instances however, we detected a strong activity (about 15-mm-diameter inhibition zone) against P. aeruginosa from the supernatant of P. tricornufum. These preparations were obtained from late stationary phases suggesting the release in older cultures of a k e e nondialysable antipseudomonad component. Using the algal growth system described by Marsot et al. (198 1a , 198 1 b ) , it was possible to corroborate previous results whereby Bacillariophyceae produced lipophilic active substances inhibiting gram-positive terrestrial bacteria and marine bacteria. The production of algal cells o n a 15-L scale also allowed for the recovery of water-soluble active components from algal cells. These findings, namely the detection of hydraphilic activity, associated with Bacillariophyceae in dialysis culture and active against staphylococcal as well as gram-negative bacteria, indicate that further research on therapeutic antibiotics produced by algae is warranted.

Acknowledgements

W e thank J. Fauteux and M. Leclerc for excellent technical assistance and C. Blais for his help in revising the manuscript. This work was partly supported by a grant (A 1297) from the Natural Sciences and Engineering Research Council of Canada. BERLAND,B . R., D. J. BONIN, A. L. CORNUS,S. Y. MAESTRINI, and J. P. MARINO. 1972. The antibacterial substances of the marine alga Stichochtysis imrnobilis (Chrysophyta).J. Phycol. 8: 383-392. BLIGH,E. G., and W. J. DYER.1959. Arapidmethodof total lipid extraction and purification. Can. J. Biochem. Physiol. 37: 911-917.

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CACCAMESE, S., and R. AZZOLINA.1979. Screening for antimicrobial activities in marine algae from eastern Sicily. Planta Med. 37: 333-339. DEBRO,L. Pi., and H. 3. WARD.1979. Antibacterial activity of freshwater green algae. Planta Med. 36: 375-378. DUFF,D. C. B., D. L. BRUCE,and N. J. ANTIA.1966. The antibacterial activity of marine planktonic algae. Can. J. Microbiol. 12: 877-884. GAUTHIER, M. J. 1980. Note sur la frCquence de la production d'antibiotiques ligidiques chez les algues planctoniques. Rev. Int. Oceanogr. Med. 58: 41 -44. GAUTHIER,M. J., P. BERNARD, and M. AUBERT.1978. Production d'un antibiotique lipidique photosensible par la diatomde marine Chaetoceros lauderi (Ralfs) . Ann. Microbiol. (Paris), 129B: 63-70. GOULD,G . W. 1969. Methods for studying bacterial spores. In Methods in microbiology. Vol. 6 A. Edited by J. R. Noms and D. W. Ribbons. Academic Press, NY. pp. 327-38 1 . Gow, J. A., I. W. DEVOE, and R. A. MACLEOD.1973. Dissociation in a marine pseudomonad. Can. J. Microbiol. 19: 695-701. GUILLARD, R. R. L., and J. H. RYTHER.1962. Studies on marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea (Cleve) Gran. Can. J. Microbiol. 8: 229-239. KOGURE,K., U. SIMIDU,and N. TAGA. 1979. Effect of Skeletonerna costaturn (Grev.) Cleve on the growth of marine bacteria. J. Exp. Mar. Biol. Ecol. 36: 201-215. MARSOT,P., R. FOURNIER, and C. BLAIS.1981a. Culture h dialyse: emploi de fibres creuses dialysantes pour la culture massive de phytoplancton. Can. J. Fish. Aquat. Sci. 38: 905-91 1 . 1981 b. Un nouveau procCd6 de culture continue a dialyse pour le phytoplancton. Biotechnol. Lett. 3: 689694.