R K Singh et al.

/ Journal of Pharmacy Research 2010, 3(9),2096-2098

Research Article
ISSN: 0974-6943 Available online through
www.jpronline.info
Screening of cyanobacterial extracts against bacteria causing nosocomial infections
R K Singh1 , S Upadhyay1 , S P Tiwari 2 , A K Rai3 and T M Mohapatra1*
1
Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi- 221005, India.
2
Department of Microbiology, VBS Purvanchal University, Jaunpur-222001, India.
3
Department of Botany, Banaras Hindu University, Varanasi- 221005, India
Received on: 15-05-2010; Revised on: 12-06-2010; Accepted on:19-08-2010

ABSTRACT
Cyanobacteria are being recognized as a potent source of bioactive secondary metabolites with antimicrobial activities during last two decades. Aqueous and organic
extracts of six locally isolated freshwater cyanobacterial strains were assessed for its antibacterial activity against three reference bacterial strains. The methanol
extract of a unicellular strain, showing largest zones of growth inhibition were found to be active against bacteria causing nosocomial infections. Antibacterial activity
was based on the agar well diffusion and agar dilution assays. Absorption maxima value of active fraction of this extract indicates the presence of Kawaguchipeptin B,
a cyclic undecapeptide and other compounds in it. Minimum inhibitory concentration (MIC) values suggest that the active compound may be a valuable antibiotic
candidate for treatment of nosocomial infections in human beings. To the best of our knowledge it is the first report on antibacterial activity of Microcystis
aeruginosa against Gram negative nosocomial pathogens.

Key words: Cyanobacteria, Microcystis, Antibacterial, Nosocomial infection.

INTRODUCTION

Any infection that appears 48 hours or more after hospital admission or within 30
days after discharge are considered as nosocomial infection or hospital acquired
infection or health care associated infection. These infections result in increased
morbidity, mortality and duration of hospitalization worldwide [1]. According to an
estimate, 2 million patients acquire nosocomial infections which cost an average
of $4.5 billion to $11 billion annually in United States[ 2 ]. Common bacterial
pathogens causing nosocomial infections are Staphylococcus aureus and members
of family Enterobacteriaceae. In recent years, WHO has noticed that appearance
of new micro-organisms and increasing bacterial resistance are the important
factor for continuous rise in nosocomial infections.
The emerging resistant bacterial strains can be treated by new antibiotics. Unfor-
tunately, the discovery rate of new antibiotics from traditional sources like actino-
mycetes is very low in current scenario [3]. Cyanobacteria, a group of Gram nega-
tive oxygenic phtosynthetic bacteria are a newly emerging source of antibiot-
ics[4,5,6]. Medicinal potentials of cyanobacteria were first recognized in 1500 BC
when Nostoc sp. was used to treat gout, fistula and several forms of cancer[7]. They
use various systems, particularly non-ribosomal peptide synthatase (NRPS) and
polyketide synthatase (PKS) systems to produce novel natural products with
therapeutic potential[8]. They may be a preferred source of antibiotics due to their
economical cultivation[9]. Several studies reporting the antibacterial activity of
cyanobacterial extracts have been performed during last decade [10,11,12]. In present
investigation, we have screened this new microbial source for their antibacterial
property against bacterial pathogens causing nosocomial infections.
MATERIAL AND METHODS
Test Organisms
The reference bacterial strains, i.e., Staphylococcus aureus ATCC 25923, Es-
cherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853 were
obtained from culture collection. Bacterial strains, i.e., Proteus vulgaris, Citrobacter
freundii, Acinetobacter baumannii and MDR strains of Escherichia coli (BHU01
& BHU02) were isolated locally from samples of inward (ICU) patients of SS
Hospital BHU and its sensitivity pattern against stanadard antibiotics were tested
using CLSI guidelines 2006[13]. The positivity for the production of extended
spectrum -lactamase (ESBL), AmpC -lactamase (AmpC BL) and metallo -
lactamase (MBL) enzymes by bacterial strains were tested by the methods of CLSI
2005 [14].
Cyanobacterial strains
Six different cyanobacteria were isolated from water samples of ponds situated in
Varanasi city by standard microbiological techniques. These isolates were identi-
fied on the basis of their morphological features and available literature[15]. All the
cyanobacterial isolates were grown in a culture room maintained at 28+1C illumi-
nated with fluorescent lamps under 16:8h light dark period using BG-11 media[16].
The cultures were harvested in late stationary phase of growth by centrifugation at
10,000 rpm for 15 minutes and subsequently lyophilized.
Preparation of cyanobacterial extracts
The dried powdered biomass was extracted with different solvents by freeze thaw
method using different solvents (water, methanol and hexane). The mixture was
centrifuged at 10,000 rpm for 15 minutes at 40C. The supernatants were collected
and lyophilized and dissolved in deionized water to make the desired concentra-
tion.
Antibacterial assay
Qualitative antibacterial assays were performed by agar well diffusion method[17].
Bacterial lawns were prepared by spreading the bacterial suspension (1.5x108 cfu/
ml) on the surface of Mueller Hinton agar plates by using sterile cotton swabs. The
hollow wells of 8 mm diameter were created in plates by using sterile agar borer.
Extracts/ active fraction dissolved in distilled water (100 mg/ml) were directly
applied to the wells in different volume (20-100l). Control wells received only
the distilled water. Plates were then incubated at 370C for 18 h. After incubation
the zones of growth inhibition were measured manually on a millimeter scale.
The minimum inhibitory concentration (MIC) was determined by agar dilution
method as described in the CLSI guidelines[18]. The lowest concentration of extract
that inhibited bacterial growth was considered to be the MIC.
Determination of relative percentage inhibition
The relative percentage inhibition with respect to positive control was calculated
by using the following formula[19]:
Relative percentage inhibition of the test extract = 100 ( a -b)
(c-b)
Where,
a: total area of inhibition of the test extract
b: total area of inhibition of the solvent
c: total area of inhibition of the standard drug

*Corresponding author. Fractionation of extract showing antibacterial activity

T. M. Mohapatra, Professor of Microbiology, Methanol extracts of M. aeruginosa were subjected to thin layer chromatography
Institute of Medical Sciences, (TLC) for separation of antibacterial fraction. TLC plates were coated with a thin
Banaras Hindu University, Varanasi- 221005 layer of silica gel-60. The solvent used for the development of chromatogram was
Fax: 91 542 2367568 the mixture of carbon tetrachloride and methanol (9:1). The bands were visualized
Tel.: 91 542 2307516
E-mail:tmmohapatra2000@gmail.com
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 R K Singh et al. / Journal of Pharmacy Research 2010, 3(9),2096-2098
in a UV transilluminator and the position of bands was marked. The bands were Table 4. Antibacterial potential of methanol extract of M. aeruginosa.
scratched from TLC plates, dissolved in methanol and scanned for its absorption
maxima by using UV spectrophotometer. For further testing, methanol was evapo- Target bacteria The size of zone inhibition (mm) at the disk content of
40 l 60 l 80 l 100 l Gentamicin
rated to dryness and residue was redissolved in deionized water. = 4 mg = 6 mg = 8 mg = 10 mg 10 mcg

E. coli ATCC 25922 14 17 19 20 25

RESULTS S. aureus ATCC 25923 08 11 13 15 19
The different Cyanobacteria isolated in this study belong to genera Anabaena, P. aeruginosa ATCC 27853 ND 09 12 13 19
Nostoc, Cylindrospermum, Phormidium and Microcystis. The morphological fea- E. coli BHU01 ND 11 13 16 -
E. coli BHU02 ND 11 13 16 -
tures of these isolates are given in Table 1. P. vulgaris ND 12 15 17 22
Table 1. Cyanobacterial isolates and their morphological characters. C. frundii ND ND 12 14 19
A. baumanii ND ND 08 10 15
Organism Morphological features

Anabaena doliolum
Nostoc sp.
Nostoc spongiformae Thallus mucilaginous, yellowish blue-green in color. Filamenats
Cylindrospermum musicola
Phormidium fragile
Microcystis aeruginosa
Thallus mucilagenous, blue-green in color. Filaments spiral Figure1. Relative percentage inhibition of target bacteria by Microcystis
with slightly tapering ends, Average 80 cells with 5 heterocysts aeruginosa methanol extract.
in a filament. Heterocysts intercalary, barrel shaped,
5- 6 broad and 6-9 long. Vegetative cells barrel shaped,
cells constricted at cross walls.
Thallus frothy, gelatinous,Trichomes 4-8 broad without sheath.
Cells spherical, mostly with gas vacuoles. Heterocysts
ellipsoidal, 5-8 broad and 6-9 long.
long, loosely entangled, 3- 3.5 broad. Vegetative cells barrel
shaped, 2-3 long. Heterocysts spherical, heteocysts occur
intercalary at interval of 12 vegetative cells,
transverse division occurs in vegetative cells.
Thallus mucilaginous, blue-green in color, occurred in stagnant
water in bloom form. Twenty celled filament with terminal heterocysts
at both ends, cells rectangular, 3.9-4.5 m broad 5.2-6.5 long,
constricted at cross walls. Filaments with consistent mucilage.
Thallus mucilagenous, brownish blue-green in color, Sheath
diffluent. Trichomes entangled or nearly parellel.
Mucilagenous bluish green colonies with irregular shape.
Spherical cells of 4-6 in diameter. The cells divide in
three successive planes lying perpendicular to each other.

Figure 2. Minimum inhibitory concentration (MIC) of Microcystis

The results of qualitative antibacterial assay against three reference bacterial
aeruginosa methanol extract against target bacteria.
strains for different extracts of the isolated cyanobacteria are shown in Table 2.
Table 2. Antibacterial activity of different cyanobacterial extracts.
Cyanobacterial strains Extraction Antibacterial activity against
solvent S. aureus E. coli P. aeruginosa
ATCC 25923 ATCC25922 ATCC 27853

Anabaena doliolum Water - - -

Methanol - - -
Hexane - - -
Nostoc sp. Water - - -
Methanol + + -
Hexane - - -
Nostoc spongiformae Water - - -
Methanol - - -
Hexane - - -
Cylindrospermum musicola Water - - -
Methanol - + -
Hexane - - -
Phormidium fragile Water - - -
Methanol - - -
Hexane - - -
Microcystis aeruginosa Water - - -
Methanol + + + BHU 01&02 were resistant to all first, second and third line antibiotics except
Hexane - + -
imipenem. These two strains also produce different -lactamase enzymes. A.
baumannii and C. freundii were resistant to some first line antibiotics (Table
Out of 18 different extracts tested, four exhibited the antibacterial activity against
3).The relative percentage inhibition and MIC values of M. aeruginosa methanol
at least one reference strain. No any extract was found to be active against all the
extract in different bacterial bioassays are shown in Figures 1& 2. The lowest MIC
three reference bacterial strains except methanol extract of M. aeruginosa.
value was 2 mg/ml against E. coli ATCC 25922.
The antibiotic sensitivity pattern of target bacteria revealed that all three refer-
ence strains as well as P. vulgaris were sensitive to all first line antibiotics. E. coli
Thin layer chromatographic separation of methanol extract of M. aeruginosa
Table 3. Sensitivity pattern of bacterial targets against standard antibiotics resulted in five bands. Absorption maxima values of these five bands are given in
Table 5. Out of these five bands, only the fourth band was found to be active
Bacterial strains Sensitive to Resistant to against E. coli ATCC 25922.

E. coli ATCC 25922 All first line antibiotics - DISCUSSION

E. coli BHU 01 (ESBL+ve, Imipenem All first, second and third line antibiotics In last two decades, cyanobacteria have been proved a prolific source of new
AmpC BL +ve)
E. coli BHU 02 (MBL+ve) Imipenem All first, second and third line antibiotics
bioactive compounds having pharmacological significance[20,21]. These active com-
S. aureus ATCC 25923 All first line antibiotics - pounds exhibit a wide range of bioactivities that may be relevant to the natural
P. aeruginosa ATCC 27853 All first line antibiotics - environment such as antibacterial, antifungal, antiviral & cytotoxic activities and
P. vulgaris All first line antibiotics - sometimes immunomodualtion & protease inhibition activities which are not
C. frundii Tobramycin, Ciprofloxacin, Ampicillin, Cefazolin, Cefuroxime related to ecological significance. Cyanobacteria of freshwater habitats have rarely
Gentamicin
A. baumanii Carbenicillin, Gentamicin, Ceftazidine, Amikacin
been explored for their antibacterial potential. Therefore, in the present investi-
Tobramycin, Norfloxacin

Journal of Pharmacy Research Vol.3.Issue 9.September 2010 2096-2098

 R K Singh et al. / Journal of Pharmacy Research 2010, 3(9),2096-2098
gation we have isolated cyanobacterial strains from freshwater ponds situated in
Varanasi city (Lat. 25.20 N & Long. 83.00 E), India. The six cyanobacterial
species included in this study belong to Nostocaceae, Oscillatoriaceae and
Chroococcaceae families.
Among 18 cyanobacterial extracts tested, 04 (22%) exhibited antibacterial activ-
ity against at least one pathogen. These results showed that this new microbial
group has enough potential to produce antibacterial substances. Our results are in
good agreement with the observations by Jaki et al.[22]. The antibacterial activities
of extracts prepared with different solvents in our study indicate that methanol is
a good solvent for extraction of antimicrobial compounds from cyanobacterial
biomass.
The methanol extract of M. aeruginosa was found to be active against several
Gram negative nosocomial isolates. These results are contradictory with the
findings of other studies. Kreitlow et al.[23] assessed hydrophilic and lipophilic
extracts of eleven freshwater cyanobacterial strains but none of them showed
activity against Gram-negative bacteria. Martins et al.[24] observed that out of 36
extracts prepared from nine cyanobacterial strains, no one was active against
Gram negative bacteria. The active extract showed highest relative percentage
inhibition against E. coli ATCC 25922 (80%) and lowest against A. baumannii.
Thus, among the bacterial strains employed in our study, E. coli ATCC 25922 has
proven the most sensitive target for antibacterial activity.
The lowest MIC value of M. aeruginosa (methanol extract) against gram nega-
tive nosocomial isolates is 2 mg/ml. On the basis of literature[25], we can say that
the lowest MIC value of active compound (purified from this extract) will range
from 5 g-25g (0.25% of crude extract). Futhermore, the extract was found to
be active against Acinetobacter baumanii [26] and Citrobacter freundii [27], the
prevalent pathogens in nosocomial infections. This extract was also active against
strains of E. coli, producing extended spectrum -lactamase (ESBL), AmpC -
lactamase (AmpC BL) and metallo -lactamase (MBL) enzymes, causing threat-
ening nosocomial infections worldwide [28,29,30]. Thus the purified active compound
may be a valuable antibiotic candidate for treating the nosocomial infections.
Our study is the extended form of previous study by Ishida et al.[31], in which they
have purified an antibacterial compound, Kawaguchipeptin B active against S.
aureus from the methanol extract of M. aeruginosa NIES 88. Thus, to the best of
our knowledge, we are reporting the antibacterial activity of M. aeruginosa
against nosocomial isolates for the first time.
Thin layer chromatographic technique was employed to fractionate M. aeruginosa
methanol extract as TLC is the method of choice to fractionate any biological
mixture. The UV spectral property of the only antibacterial fraction indicates the
presence of Kawaguchipeptin B (absorption maxima 282 nm) in it. However,
there may be other compounds in this fraction as Kawaguchipeptin B does not act
against Gram negative bacteria. Further, the bioassay guided purification of anti-
bacterial compounds from this fraction is in progress.
ACKNOWLEDGEMENT
Dr. R K Singh is thankful to UGC, India for providing Junior Research Fellowship.
Source of support: UGC, India , Conflict of interest: None Declared
Journal of Pharmacy Research Vol.3.Issue 9.September 2010
REFERENCES
1. Weinstein RA, Nosocomial infection update. Emerg Infect Dis, 4, 1998, 41620.
2. Ricks, Germ Warfare. Ms. Magazine, 2007 4345. http://www.msmagazine. com/spring2007/
germwarfare.asp.
3. FIU Bionews, Florida International University, USA. Vol 2 No. 4, 2005
4. Mundt S, Kreitlow S, Jansen R, Fatty acids with antibacterial activity from the cyanobacterium
Oscillatoria redekei HUB051. J. Appl. Phycol, 15, 2003, 263- 267.
5. Singh S, Kate BN, Banerjee UC, Bioactive critical compounds from cyanobacteria and microalgae:
an overview. Cri Rev Biotech, 25, 2005, 73-95.
6. Raveh A, Carmeli S, Antimicrobial Ambiguines from the cyanobacterium Fischerella sp. col-
lected in Israel. J. Nat. Prod, 70, 2007, 196-201.
7. Liu XJ, Chen F, Cell differentiation and colony alteration of Nostoc flagelliforme, an edible terres-
trial cyanobacterium, in different liquid suspension culture. Folia microbial, 48, 2003, 619-626.
8. Ehernich IM, Waterbury JB, Webb EA, Distribution and diversity of natural product genes in
marine and fresh water cyanobacterial cultures and genomes. App. Env. Microbiol, 71, 2005,
7401-7403.
9. Jaspars M, Lawton LA, Cyanobacteria- a novel source for pharmaceuticals. Current Opinion in
Drug Dis. Dev, 1, 1998, 77- 84.
10. Qstenswik O, Skulberg OM, Underdal B, Hormazabal V, Antibacterial properties of extracts from
selected planktonic freshwater cyanobacteria a comparative study of bacterial bioassays. J App
Microbiol, 84, 1998, 11171124.
11. Skulberg OM, Microalgae as a source of bioactive molecules; experience from cyanophyte
research. J. App. Phycol, 12, 2000, 341-348.
12. Biondi N, Tredici MR, Taton A, Wilmotte A, Hodgson DA, Losi D et al, Cyanobacteria from benthic
mats of Antarctic lakes as a new source of bioactivities. J. Appl. Microbiol, 105, 2008, 105-115.
13. Clinical Laboratory Standard Institute, Methods for antimicrobial disk susceptibility tests for bac-
teria that grow aerobically: approved standard- Ninth edition Vol. 26 (1), M2-A9. CLSI, Wayne,
PA, USA, 2006.
14. Clinical Laboratory Standard Institute, Methods for antimicrobial disk susceptibility testing, ap-
proved standard- Fifteenth edition. CLSI, Wayne, PA, USA, 2005.
15. Desikachary TV, The Cyanophyta, ICAR, New Delhi, 1959.
16. Rippka RJ, Derulles J, Waterbury JB, Herdman M, Stanier RY, Generic assignments, strain histories
and properties of pure cultures of cyanobacteria. J Gen Microbiol. 111: 1979, 1 - 61.
17. Irobi ON, Moo-Young M, Daramola S, Antimicrobial activity of Annatto (Bixa orellana) extract.
Int J Pharmacog, 34, 1996, 87-90.
18. Clinical Laboratory Standard Institute, Methods for dilution antimicrobial susceptibility tests for
bacteria that grow aerobically: approved standard- Seventh edition Vol. 26 (2) M7-A7. CLSI,
Wayne, PA, USA, 2006.
19. Ajay KK, Lokanatha RMK, Umesha KB, Evaluation of antibacterial activity of 3,5-dicyano-
4,6-diaryl-4-ethoxycarbonyl-piperid-2-ones, Journal of Pharmaceutical and Biomedical
Analysis, 27, 2002, 837-840.
20. Moore RE, Banerjee V, Bornemann V, Caplyn FR, Chen JL, Carly DJ et al, Novel cytotoxins and
fungicides from blue-green algae and marine animals possessing algal symbionts. Pure Appl
Chem, 61, 1989, 521-524.
21. Zainuddin EN, Jansen R, Nimtz M, Wray V, Preisitsch M, Lalk M, Mundt S, Lyngbyazothrins A-
D, Antimicrobial cyclic undecapeptides from the cultured cyanobacterium Lyngbya sp. J Nat
Prod, 72, 2009, 1373-1378.
22. Jaki B, Orjala J, Brgi HR, Sticher O, Biological screening of cyanobacteria for antimicrobial and
molluscicidal activity, brine shrimp lethality, and cytotoxicity. Pharmaceutical Biol, 37, 1999, 138
143.
23. Kreitlow S, Mundt S, Lindequist U, Cyanobacteriaa potential source of new biologically active
substances. J Biotech, 70, 1999, 6163.
24. Martins RF, Ramos MF, Herfindal L, Sousa JA, Skarven K and Vasconcelos VM, Antimicrobial and
cytotoxic assessment of marine cyanobacteria - Synechocystis and Synechococcus. Mar Drugs,
6, 2008, 111.
25. Luesch H, Pangilinan R, Yoshida WY, Moore R E, Paul VJ, Pitipeptolides A and B, New
Cyclodepsipeptides from the Marine Cyanobacterium Lyngbya majuscule.J.Nat.Prod,64, 2001,
304-307.
26. Peleg AY, Seifert H, Paterson DL, Acinetobacter baumannii: Emergence of a successful Pathogen.
Clin Microbiol Rev, 21, 2008, 538-82.
27. Whalen JG, Mully TW, Enlgish JC 3rd , Spontaneous Citrobacter freundii infection in an immuno-
competent patient. Archives of Dermatology, 143, 2007, 124-125.
28. Emery CL, Weymouth LA, Detection and clinical significance of extended-spectrum beta-
lactamases in a tertiary-care medical center. J Clin Microbiol, 35, 1997, 20612067.
29. Philippon A, Arlet G, Jacoby GA, Plasmid-determined AmpC-type -lactamases, Antimicrob
Agents Chemother 46, 2002, 111.
30. Walsh TR, Toleman MA, Poirel L, Nordmann P, Metallo-beta-lactamases: the quiet before the
storm? Clin Microbiol Rev, 18, 2005, 306-325.
31. Shida K, Matsuda H, Murakami M and Yamaguchi K, Kawaguchipeptin B, an antibacterial cyclic
undecapeptide from the cyanobacterium Microcystis aeruginosa. J Nat. Prod, 60, 1997, 724-726.
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