Cah. Biol. Mar.

(2012) 53 : 45-51

Antimycobacterial activity of marine cyanobacterial species

against Mycobacterium smegmatis

Murugesan CHANDRASEKARAN1 and Muthuraman SUNDARARAMAN2*

(1)
Department of Plant Science, Bharathidasan University, Tiruchirrappalli -620 024, Tamilnadu, India
(2) Department of Marine Biotechnology, Bharathidasan University, Tiruchirappalli -620 24, Tamilnadu, India
*Corresponding author: Fax: +91 431 2407084, E-mail: sundarbdu1@gmail.com

Abstract: The studies of antimycobacterial activity of extracts (ethanol, ethyl acetate and hexane) from 18 marine
cyanobacterial strains have shown specific inhibitory activity against Mycobacterium smegmatis. All the strains were
evaluated for antimycobacterial activity by disc diffusion method followed by spread plate, broth dilution and streak plate
methods. It was found that among the 18 cyanobacterial species, Oscillatoria laetevirens BDU 141071 had the highest
activity in all the three solvent systems. Among them three strains, Oscillatoria willei BDU 130711, Oscillatoria willei
BDU 130791 and Phormidium corium BDU 60201 had no activity. The results revealed that most of the ethyl acetate
extracts (13 cyanobacterial strains) were active against Mycobacterium smegmatis compared to ethanol (eight cyano-
bacterial strains) and hexane (five cyanobacterial strains) extracts. The present study shows that some marine cyanobacteria
harbor potential lead compounds having antimycobacterial activity.

Résumé : Activité antimycobactérienne de cyanobactéries marines contre Mycobacterium smegmatis. Les études d’activité
antimycobactérienne d’extraits (ethanol, ethyl acetate and hexane) de 18 souches de cyanobactéries marines ont montré une
activité inhibitrice spécifique contre Mycobacterium smegmatis. Toutes les souches ont été évaluées pour l’activité anti-
mycobactérienne par la méthode de diffusion de disque suivie par la plaque de propagation, la dilution de bouillon et les
méthodes de plaque latérale. Parmi les 18 espèces de cyanobactéries, Oscillatoria laetevirens BDU 141071 avait la plus
forte activité dans les trois types de solvant. Trois souches, Oscillatoria willei BDU 130711, Oscillatoria willei BDU
130791 et Phormidium corium BDU 60201 n’ont montré aucune activité. Les résultats ont révélé que la plupart des extraits
d’acétate d’éthyle (13 souches) étaient actifs contre Mycobacterium smegmatis, davantage qu’à l’éthanol (8 souches) et à
l’hexane (5 souches). La présente étude montre que quelques cyanobactéries marines recèlent des composés potentielle-
ment majeurs pour l’activité antimycobactérienne.

Keywords: Marine Cyanobacteria l Antimycobacterial compounds l Mycobacterium smegmatis l Blue green algae

Reçu le 24 septembre 2010 ; accepté après révision le 11 août 2011.

Received 24 September 2010; accepted in revised form 11 August 2011.
46 ANTIMyCOBACTERIAL ACTIVITy OF MARINE CyANOBACTERIA

Introduction cyanobacterial strains with potential activities against

multi-drug resistant M. tuberculosis.
The demand of therapeutic drugs from natural resources is
on the increase in the 21st century (Borowitzka, 1995; Materials and methods
Mundt et al., 2001; Ramamurthy & Raveendran, 2009; Al-
Haj et al., 2010; Villa & Gerwick, 2010) and the
contribution of marine organisms is potentially remarkable Bacterial strain and growth condition
(Sponga et al., 1999; Jha & Zi-rong, 2004; Bérdy, 2005; Culture of Mycobacterium smegmatis was obtained from
Berrue et al., 2007; Wei et al., 2007; Jain & Sonawane, Tuberculosis Research Center (TRC), Chennai, Tamilnadu,
2008). Cyanobacteria (also known as blue–green algae) are India. This standard strain was confirmed with
a group of unusually diverse Gram-negative photoau- morphological and biochemical analysis. Culture
totrophic prokaryotes that originated 3.5 billion years ago. suspensions were prepared from Middle Brook 7H9 broth
Various strains of cyanobacteria are known as producing and cultures were maintained on Middle Brook 7H10 agar
intracellular and extracellular metabolites with diverse bio- medium containing 0.5% glycerol and 10% ADC
logical activities such as antialgal, antibacterial, antifungal, enrichment, incubated at 37°C for 2 days.
antiviral, anthelmintic, and antiprotozoal activity (Issa,
1999; Burja et al., 2001; Tan, 2007; Mayer et al., 2007, Cyanobacterial strains and biomass
2009 & 2011; Patra et al., 2009). They have tremendous
potential to be used as therapeutic agents for a variety of Marine cyanobacterial strains (Table 1) were obtained from
diseases (Shibib et al., 1993; Patterson et al., 1994, the Germplasm of National Facility for Marine
Romanos et al., 2002). Secondary metabolites from Cyanobacteria (NFMC), Bharathidasan University,
cyanobacteria have also exhibited toxic, hormonal, cyto- Tiruchirappalli, Tamilnadu, India. The cyanobacterial
toxic and antineoplastic activities (Carmichael, 1992; strains were grown in ASN-III (marine synthetic) medium
(Rippka et al., 1979). The marine cyanobacterial cultures
Patterson et al., 1994; Harada et al., 2002; Tan, 2007;
were inoculated into 250 mL Erlenmeyer flask containing
Mayer et al., 2007 & 2011).
100 mL of sterile ASN-III media and incubated under white
Tuberculosis (TB) is the leading cause of mortality all
fluorescent light 13.7 µE.m-2.s-1 at 27 ± 2°C with 14/10 hr
over the world due to infectious agent Mycobacterium
L/D cycle. After incubation, cultures were harvested at
tuberculosis (Wallis, 1996; Ryan & Ray, 2004). WHO
stationary phase (20 days) and centrifuged at 10,000 rpm
(2010) reported that more than 2 billion people equal to
for 20 min.
one-third of the world’s population are infected with
Mycobacterium tuberculosis. The vast majority of TB
deaths occur in the developing world and more than half of Table 1. List of Marine cyanobacterial strains used for anti-
the deaths in Asia (WHO, 2010). There were 9.4 million mycobacterial studies.
new TB cases in 2008 (3.6 million of whom are women) Tableau 1. Liste des souches cyanobactériennes marines
including 1.4 million cases among them infected with HIV. utilisées pour les tests antimycobactériens.
During 2008, 1.8 million people have died due to TB and it Serial No. Name of the Organism
indicates 4500 deaths a day due to Multidrug-Resistant TB
(MDR-TB) (WHO, 2010). The resistance to second-line 1. Gleocapsa crepidium BDU 20121
drugs developed on top of MDR-TB leads to extensively 2. Phormidium corium BDU 60201
3. Phormidium valderianum BDU 41001
Drug-Resistant TB (XDR) (WHO, 2010). The worldwide
4. Phormidium valderianum BDU 140041
problem caused by TB and the lack of new drugs in the 5. Phormidium valderianum BDU 142552
market trigger an imperative search for novel lead 6. Phormidium valderianum BDU 142022
molecules to fight efficiently against the rapid spread of 7. Oscillatoria boryana BDU 40261
multidrug-resistant TB (Copp, 2003). In this context, there 8. Oscillatoria formosa BDU 130511
is an urgent need for new anti-TB drugs with less toxic side 9. Oscillatoria laetevirens BDU 100891
effects, improved pharmacokinetic properties, extensive 10. Oscillatoria laetevirens BDU 141071
and potent activity against resistant strains. 11. Oscillatoria salina BDU 92021
12. Oscillatoria willei BDU 130711
Marine cyanobacteria have been recognized as an
13. Oscillatoria willei BDU 130791
important renewable bioresource for novel bioactive 14. Lyngbya sp.
compounds. We investigated the antimycobacterial activity 15. Lyngbya sp. BDU 30342
of the organic solvent extracts from 18 marine 16. Lyngbya sp. BDU 140301
cyanobacteria, as a pilot screening study by employing M. 17. Spirulina subsalsa BDU 141021
smegmatis which is a suitable surrogate screen for selecting 18. Nostoc calcicola BDU 40302
 M. CHANDRASEKARAN, M. SUNDARARAMAN
Isolation of antimycobacterial metabolites
To study the antimycobacterial activity, cyanobacterial
extracts were derived (ethanol, ethyl acetate and hexane) by
solvent extraction method (Shibib et al., 1993).
Cyanobacterial biomass was ground with ethanol (1:2 w/v)
and glass powder. The macerate was kept at 4ºC for 12 hrs.
After incubation, the supernatant was separated. The
deposit was re-subjected to grind with ethanol in the ratio
of 1:1 (w/v), until it became pale in color; it was incubated
at room temperature for 3 hrs. The pooled supernatant was
concentrated with feed back vaccum concentrator (Savant,
USA). The same procedure was used to prepare extracts
from ethyl acetate and hexane solvents. The concentrated
extracts were dissolved in DMSO (Dimethyl sulfoxide).
The reconstituted (DMSO dissolved) extracts were
examined for antimycobacterial activity against
Mycobacterium smegmatis.
Determination of the antimycobacterial activity
Disc diffusion assay. Antimycobacterial activity was
evaluated by disc diffusion technique (Bauer et al., 1966).
Sterile filter paper discs having 6 mm of diameter were
prepared by pipetting 15 μL of extract to each disc and then
the discs were placed on Middle Brook 7H10 agar plates.
After incubation for 72 hours at 37°C, a clear zone around
a disc was evidence of antimycobacterial activity. Diameter
of the zone of inhibition was measured in millimeters. Each
test was prepared in duplicate; discs loaded with the
extracting agents were tested as controls.
Spread Plate Method. For the spread plate technique, the
inoculum (100 µl of mycobacterial culture and 100 µL of
ethyl acetate) was spread evenly over the entire surface of
the Middle Brook 7H10 agar. The plates spread with
DMSO (100 µL of mycobacterial culture and 100 µL of
DMSO) and test organism (100 µL of mycobacterial
culture and 100 µL of Middle Brook 7H9 broth) were
treated as controls. The culture was incubated at 37°C for
72 hours. After incubation, the extracts added plates
compared with controls.
Broth Dilution Method. The cyanobacterial extracts which
showed inhibitory activity both in disc diffusion and spread
plate method was further investigated using the broth
dilution method. The experimental set up was made as
follows (i) 1 mL of broth as control with 900 µL broth and
100 µL of Mycobacterium smegmatis (organism control);
(ii) 800 µL broth with 100 µL culture, 100 µL DMSO
(DMSO control) and (iii) 800 µL broth with 100 µL culture,
100 µL ethyl acetate extracts (test). The culture was
incubated for 72 hrs at 37°C. After incubation the results
were observed for further confirmation.
Streak Plate Method. The confirmation of inhibitory
47
activity in broth dilution method was made by performing
streak plate method. A loopful of M. smegmatis culture was
streaked uniformly over the Middle Brook 7H10 agar
which contains 100 μL of extracts. In this technique, culture
alone on the plate acted as organism control and DMSO as
solvent control. The plates were incubated at 37°C for 72
hrs. All the experiments were conducted in triplicate. The
results were observed and interpreted.
Results and discussion
The antimycobacterial activity of crude extracts of 18
marine cyanobacterial strains were evaluated by disc
diffusion method and the results were summarized in Table
2. Among 18 marine cyanobacterial strains subjected for
primary screening process, all of them have shown activity
against M. smegmatis, except three isolates (Oscillatoria
willei BDU 130711, Oscillatoria willei BDU 130791 and
Phormidium corium BDU 60201). Hence, the
cyanobacteria showed antimycobacterial activity were
considered for further investigation.
Among 15 marine cyanobacterial extracts, Oscillatoria
formosa BDU 130511, Phormidium valderianum BDU
140041, Oscillatoria boryana BDU 40261 showed activity
only in ethyl acetate extract (Table 2 & Fig. 1). There was
no activity observed in ethanol and hexane extracts. The
zone of inhibition is less than 2 mm indicating minimum
antimycobacterial activity in certain isolates.
Ethanolic extracts of Oscillatoria salina BDU 92021
and Phormidium valderianum BDU 142552 exhibited
reasonable activity, but there was no activity in ethyl
acetate and hexane extracts. As the zone of inhibition was
below 2 mm the compounds showed least activity.
Nostoc calcicola BDU 40302, Spirulina subsalsa BDU
141021, Gleocapsa crepidium BDU 20121, Lyngbya sp.
BDU 30342 and Lyngbya sp. BDU 140301 have indicated
higher activity (above 2 mm) only in ethanol and ethyl
acetate extraction. Among them, Lyngbya sp. BDU 30342
exhibited good activity in both ethanol and ethyl acetate
extraction than other cyanobacterial strains.
Both ethyl actate and hexane extracts of Phormidium
valderianum BDU 41001, Phormidium valderianum BDU
142022, Lyngbya sp. and Oscillatoria laetevirens BDU
100891 (Table 2) have exhibited antimycobacterial activity,
but not in ethanolic extracts.
Of all other cyanobacterial species tested, only
Oscillatoria laetevirens BDU 141071 has shown activity
against Mycobacterium smegmatis effectively (Table 2 &
Fig. 1). In addition to that, it showed activity in all the three
different solvent systems used in this study. The zone of
inhibition was above 2 mm, indicating that some bioactive
metabolites present in this organism showed antimyco-
bacterial activity. In the previous studies, the variation of
48 ANTIMyCOBACTERIAL ACTIVITy OF MARINE CyANOBACTERIA

Table 2. Disc diffusion assay for the cyanobacterial extracts.

Tableau 2. Analyse de diffusion de disque pour les extraits cyanobactériaux.

Serial No. Name of the Organism Ethanol Extracts Ethyl acetate extracts Hexane extracts

1. Disc Control - - -
2. Solvent control - - -
3. DMSO control - - -
4. Gleocapsa crepidium BDU 20121 + ++ -
5. Phormidium corium BDU 60201 - - -
6. Phormidium valderianum BDU 41001 - ++ ++
7. Phormidium valderianum BDU 140041 - ++ -
8. Phormidium valderianum BDU 142552 + - -
9. Phormidium valderianum BDU 142022 - + ++
10. Oscillatoria boryana BDU 40261 - ++ -
11. Oscillatoria formosa BDU 130511 - ++ -
12. Oscillatoria laetevirens BDU 100891 - ++ ++
13. Oscillatoria laetevirens BDU 141071 ++ ++ ++
14. Oscillatoria salina BDU 92021 + - -
15. Oscillatoria willei BDU 130711 - - -
16. Oscillatoria willei BDU 130791 - - -
17. Lyngbya sp. - ++ ++
18. Lyngbya sp. BDU 30342 ++ ++ -
19. Lyngbya sp. BDU 140301 + ++ -
20. Spirulina subsalsa BDU 141021 + ++ -
21. Nostoc calcicola BDU 40302 + ++ -

- No activity; + ≤ 4 mm (zone of inhibition); ++ ≥ 5 mm (zone of inhibition).

Figure 1. Disc Diffusion assay of three organic solvent

extracts.
Figure 1. Analyse de diffusion de disque de trois extraits de
dissolvants organiques.
antibacterial activity of the organic solvent extracts was due
to the presence of different antibacterial substances among
these species (Schwartz et al., 1990; Patterson et al., 1994;
Newman & Cragg, 2007; Rania & Hala, 2008; Mathivanan
et al., 2010).
Based on the experimental results, among 18 cyano-
bacterial species, four organisms (Nostoc calcicola BDU
Figure 2. Spread plate method for Mycobacterium smegmatis
against the selected ethyl acetate extracts from marine
cyanobacteria S1-Nostoc calcicola BDU 40302, S12-Lyngbya sp.
BDU 30342, S14-Lyngbya sp. BDU 140301, S16-Oscillatoria
laetevirens BDU 141071.
Figure 2. Méthode de plaque de propagation pour
Mycobacterium smegmatis contre les extraits d’acétate éthylique
des cyanobactéries marines S1-Nostoc calcicola BDU 40302,
S12-Lyngbya sp. BDU 30342, S14-Lyngbya sp. BDU 140301,
S16-Oscillatoria laetevirens BDU 141071.
 M. CHANDRASEKARAN, M. SUNDARARAMAN 49

Table 3. Status of Mycobacterium smegmatis growth against the selected ethyl acetate extracts from marine cyanobacteria.
Tableau 3. Statut de croissance de Mycobacterium smegmatis contre les extraits choisis d’acétate éthylique du marine cyanobacteria.

Status of Mycobacterium smegmatis growth

Name of the Organism Spread plate method Broth dilution method Streak plate method

Organism control + + +
DMSO control + + +
Nostoc calcicola BDU 40302 (S1) - - -
Lyngbya sp. BDU 30342 (S12) - - -
Lyngbya sp. BDU 140301(S14) - - -
Oscillatoria laetevirens BDU 141071 (S16) - - -

+ = Growth appeared; - = Not grown

Figure 3. Broth dilution method for Mycobacterium

smegmatis against the selected ethyl acetate extracts from marine
cyanobacteria T1-Broth Control, T2-Culture Control, T3-S1-
Nostoc calcicola BDU 40302, T4-S12-Lyngbya sp. BDU 30342,
T5-S14-Lyngbya sp. BDU 140301, T6-S16-Oscillatoria laete-
virens BDU 141071, T7 - DMSO control.
Figure 3. Méthode de dilution de bouillon pour
Mycobacterium smegmatis contre les extraits d’acétate d’éthyle
des cyanobactéries marines T1-Broth Control, T2-Culture témoin,
T3-S1-Nostoc calcicola BDU 40302, T4-S12-Lyngbya sp. BDU
30342, T5-S14-Lyngbya sp. BDU 140301, T6-S16-Oscillatoria
laetevirens BDU 141071, T7 - DMSO témoin.
40302, Lyngbya sp. BDU 30342, Lyngbya sp. BDU 140301,
Oscillatoria laetivirens BDU 141071) showed higher
activity. These organisms are subjected to further studies.
In spread plate method, (Table 3 & Fig. 2) the growth
was observed in organism control and DMSO control.
Whereas in cyanobacterial extracts seeded plate, there was
no growth, which may indicate that cyanobacterial
compounds have had some antimycobacterial activity
against Mycobacterium smegmatis.
In broth dilution method (Table 3 & Fig. 3), complete
growth inhibition was observed in extract added tubes.
Whereas, the growth was observed in both organism and
DMSO controls. Though extract added tubes showed some
turbidity, which is not due to the growth of mycobacterium.
This turbidity may be attributed to the reaction between
compounds present in the extracts and broth.
This was further confirmed through streak plate method
(Table 3 & Fig. 4), in which organism and DMSO controls
Figure 4. Streak plate method for Mycobacterium smegmatis
against selected cyanobacterial extracts from marine
cyanobacteria S1-Nostoc calcicola BDU 40302, S12-Lyngbya sp.
BDU 30342, S14-Lyngbya sp. BDU 140301, S16-Oscillatoria
laetevirens BDU 141071.
Figure 4. Méthode de plaque latérale pour Mycobacterium
smegmatis contre les extraits des cyanobactéries marines S1-
Nostoc calcicola BDU 40302, S12-Lyngbya sp. BDU 30342, S14-
Lyngbya sp. BDU 140301, S16-Oscillatoria laetevirens BDU
141071.
showed profuse growth, whereas complete growth
inhibition was observed in test plates. The present work
clearly indicated the potentials of marine cyanobacterial
metabolites, which were effective against Mycobacterium
smegmatis.
For complete characterization of bioactive principles,
the crude extracts should be purified further and analyzed
for structure elucidation. Though antimycobacterial
extracts analyzed might not be considered as new
therapeutic agents, this preliminary study is found to be
promising for future investigations. For the identification of
the antimicrobial compounds, it is necessary to obtain it in
pure form employing a series of purification process and
different chemical analysis such as chromatography,
50 ANTIMyCOBACTERIAL ACTIVITy OF MARINE CyANOBACTERIA
spectroscopy and other sophisticated techniques like GC-
MS, NMR and so on. This study indicates that there is a
vast scope for marine cyanobacteria in search for anti-
mycobacterial compounds.
Acknowledgment
We thank National Facility for Marine Cyanobacteria
(NFMC), Bharathidasan University, Tiruchirappalli-620
024, Tamilnadu, India, for providing facilities and support.
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