Biotechnol Lett (2006) 28:1083–1088

DOI 10.1007/s10529-006-9054-2

ORIGINAL PAPER

Production and purification of a bioactive substance

inhibiting multiple drug resistant bacteria and human
leukemia cells from a salt-tolerant marine Actinobacterium
sp. isolated from the Bay of Bengal
Malay Saha Æ Parasuraman Jaisankar Æ Satadal Das Æ Kalyan K. Sarkar Æ
Soma Roy Æ Shila E. Besra Æ Joseph R. Vedasiromani Æ Debashish Ghosh Æ
Barindra Sana Æ Joydeep Mukherjee

Received: 23 January 2006 / Accepted: 22 March 2006 / Published online: 21 June 2006
 Springer Science+Business Media B.V. 2006

Abstract Four marine actinobacteria tolerant to
200 g NaCl l)1 were screened for antibacterial
activity against eight patient-derived multiple
drug resistant (MDR) bacteria. The active com-
pound (MW 300.2, predicted molecular formula
C20H28O2) from an actinobacterium, was inhibi-
tory to three Gram-positive and three Gram-
negative MDR bacteria, seven non-clinical
Gram-positive, four Gram-negative bacteria and
five fungi (MIC: 3.5–4.0 lg ml)1). Also, 54% of
human leukemia (HL-60) cells were killed by the
compound at 0.05 lg ml)1. Bioreactor production
demonstrated unusual primary metabolite kinet-
ics. Molecular phylogenetic analysis showed this
typical intertidal inhabitant to be a member of
the Streptomyces genus and distinct from other
salt-tolerant actinobacteria. As no compound was
M. Saha Æ D. Ghosh Æ B. Sana Æ J. Mukherjee (&)
Environmental Science Programme and Department
of Life Science & Biotechnology, Jadavpur
University, Kolkata 700 032, India
e-mail: joymukherjee@vsnl.net
P. Jaisankar Æ K. K. Sarkar Æ S. Roy Æ
S. E. Besra Æ J. R. Vedasiromani
Indian Institute of Chemical Biology, 4, Raja S.C.
Mullick Road, Kolkata 700 032, India
S. Das
Microbiology and Immunology Department, Peerless
Hospital and B.K. Roy Research Centre, Kolkata 700
094, India
found to match the properties in several elec-
tronic databases, our screening strategy should
increase the possibility of discovering bioactive
molecules from rare actinobacteria.
Keywords Actinobacteria Æ Anti-leukemic Æ
Antimicrobial Æ Bioreactor Æ Marine Æ Phylogeny
Introduction
Marine-derived antibiotics may be more efficient
at fighting infections because the terrestrial bac-
teria have not developed any resistance against
them (Donia and Hamman 2003). Marine ac-
tinobacteria have been projected as potential
sources of novel antimicrobial compounds (Bull
et al. 2005). So, we selected marine actinobacteria
that can grow in 200 g l)1 NaCl and screened the
salt-tolerant colonies against recalcitrant patient-
derived multiple drug resistant (MDR) bacteria.
The active compound was isolated and also tested
for its effect on bacteria (including MDR bacte-
ria), fungi and human leukemia cells.
Materials and methods
Isolation of actinobacteria
Sediment from the Bay of Bengal (Lat. 20
50¢ N,
Long. 88
19¢ E) was collected and actinobacteria

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were isolated in a medium (all units in g l)1):
starch 10, casein 3.0, peptone 1, malt extract 10,
K2HPO4 0.5, yeast extract 1, agar 15, distilled
water 500 ml, natural sea water 500 ml, pH 7.3
with varying concentrations of NaCl (0–
200 g l)1). Four colonies growing in 200 g
NaCl l)1 were screened for antibacterial activity
against patient-derived MDR bacteria (obtained
from the Peerless Hospital & B.K. Roy Research
Centre, Kolkata, India) as indicated in Table 1.
The four isolates were grown in the production
medium (PM, all units g l)1): starch 2, glucose 2,
soybean meal 2, yeast extract 0.5, NaCl 0.25,
CaCO3 0.32, CuSO4 0.005, MnCl2 0.005, ZnSO4
0.005, natural sea water 500 ml, distilled water
500 ml, pH 7.3 for 96 h. About 150 ll of the
culture supernatant was placed in each of the cups
of Luria–Bertani agar plates which were previ-
ously swabbed with the culture of the MDR
bacteria and incubated at 37
C. Isolate MS1/7 was
selected for further studies.
Growth
Intracellular protein of 100 ll supernatant, ob-
tained from the sonicated cell pellet, was deter-
mined by the Bradford’s reagent to record growth
at varying NaCl levels.
Production in a bioreactor
Starch and glucose were replaced by an equiva-
lent amount of maltose (which enhanced the
production of the antimicrobial compound) in the
PM. About 10 ml inoculum, grown in PM med-
ium, containing 2.5 · 105 live cells per ml was
added to 2 l of PM. The aeration rates and shaft
speed of a bioreactor (Eyela MBF, Japan) were
varied to identify the best process condition.
Sample supernatants were tested for antimicro-
bial activity and residual maltose (by reaction
with dinitrosalicylic acid). Growth was deter-
mined as above.
Isolation and purification
The pH of the culture filtrate was lowered to 4.5,
extracted with 3 vol. butyl acetate and, after
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Biotechnol Lett (2006) 28:1083–1088
separation and subsequent evaporation of the
organic phase, the residue was dissolved in
methanol and stored at 0–4
C. The crude extract
was applied to a silica gel column [chloro-
form:petroleum ether (1:19 v/v) as mobile phase].
The active fractions were pooled, dried under
vacuum to yield a brownish gum and applied to a
semi-preparative reverse phase HPLC (Shimadzu
SPD 6AV, Japan) C18 column (length 32.5 cm,
diam 1.11 cm), mobile phase pure methanol, UV
detection at 260.4 nm. The eluates with retention
time of about 3.6 min were pooled and dried.
About 20 mg of the purified antibiotic was ob-
tained from 8 l (by combining four batches of the
reactor). Mass spectrometry (ESI) was done using
Q-TOF Micromass (LCMS) spectrometer, IR
spectrum was recorded on a JASCO-FT-IR
Model 410 using samples (neat) on KBr cells and
1
H NMR (300 MHz) spectrum was recorded on a
Bruker DPX 300 NMR instrument.
Determination of minimum inhibitory
concentration (MIC)
The MIC of the purified antibiotic was deter-
mined against six MDR bacteria (Table 1) and
microorganisms mentioned in the results section
by the agar dilution method (EUCAST Definitive
Document, E.Def. 3.1, 2000). About 104 c.f.u. per
spot of the bacteria were spotted onto Mueller–
Hinton agar plates and incubated at 37
C for
18 h. The MIC values against fungi were deter-
mined following the method of Liu et al. 2002.
Freshly made fungal suspensions (104 cells ml)1)
were inoculated onto the PDA plates and incu-
bated at 26
C for 48–72 h. Inhibitory concentra-
tions were also determined by counting cell
viability after incubating the compound against
human leukemia cell line (HL-60) for 24 h. All
determinations with the pure compound were
done in the range 0–10 lg ml)1.
Toxicity
Hemolytic activity of the purified compound to-
wards murine and human erythrocytes was tested
by the method reported in our earlier article
(Saha et al. 2005).
Biotechnol Lett (2006) 28:1083–1088 1085

Characterization of the producing Results and discussion

microorganism
The biomass of the isolate MS1/7 was washed
twice in sterile Tris/EDTA buffer and approxi-
mately 100 mg was used for DNA extraction. The
purification of DNA, amplification and sequenc-
ing of the 16S rRNA gene were carried out as
described (Imtech Laboratory Manual on Ac-
tinomycetes 1998). Forward primer, 5¢-CAG GCC
TAA CAC ATG CAA GTC-3¢ and reverse pri-
mer, 5¢-GGG CGG WGT GTA CAA GGC-3¢
(Marchesi et al. 1998) were selected for PCR
amplification (Alm et al. 1996). The composition
of the PCR reaction mixture, PCR cycle, purifi-
cation and sequencing of the amplified DNAs and
phylogenetic analysis of the sequence were done
as described in our earlier publications (Saha et al.
2005; Sana et al. 2006a, b). A phylogenetic tree
was constructed by using the neighbour joining
DNA distance algorithm (Saitou and Nei 1987).
Antimicrobial and cytotoxic activity
In accordance to results in Table 1, MS1/7 was
selected for further studies. MIC values of the
purified compound of this isolate against six
MDR bacteria (Table 1) and Bacillus pumilis
MTCC 1607, Mycobacterium smegmatis MTCC
06, Staphylococcus aureus MTCC 96, Bacillus
subtilis MTCC 441, Arthrobacter protophormiae
MTCC 2682, Lactobacillus lactis MTCC 3038,
Micrococcus luteus MTCC 106, Pseudomonas
aeruginosa MTCC 2453, Serratia marcescens
MTCC 86, Escherichia coli DH 5(a, Proteus
mirabilis MTCC 425, Poria pruinosum MTCC
412, Aspergillus niger MTCC 1344, Candida
albicans MTCC 227, Saccharomyces cerevisae
MTCC 170 and Neurospora crassa MTCC 416
were in the range 3.5–4 lg ml)1 indicating that
the compound is highly active against a broad

Table 1 Antimicrobial activity of the four salt tolerant Actinobacteria (MS1/7, MS2/7, MS3/7, MS4/7) against multiple drug
resistant bacteria and MIC values of the purified compound of MS1/7
Multiple drug resistant bacteria Zone of inhibitiona MICb (lg ml)1)
MS 1/7 MS 2/7 MS 3/7 MS 4/7

Escherichia coli (Diagnostic Serial 12134) 20 – 16 14 4.0

(A2, A3, C3, C5, C9, C10, C12, D, K N1, N3, O2)
c
Escherichia coli (Diagnostic Serial 12426) 21 – 13 –
(A2, A3, C12, C5, N1)
Salmonella enterica (Diagnostic Serial 7211044438) 24 11 – 13 3.5
(A4, C2, C4, C8, C9, C10, G, O1, P, S)
c
Staphylococcus aureus (Diagnostic Serial 721069794) 19 14 11 16
(A1, C1, C10, G, N2, O1, P)
MRSA (Diagnostic Serial 23602) 23 – – – 4.0
(C10, C1, C12, M, N3, O2)
MRSA (Diagnostic Serial 23411) (A4, C1, C7, C10, 21 – 14 – 4.0
C11, C12, M, N3, O1)
MRSA (Diagnostic Serial 13245) 18 15 – 11 3.5
(A4, C2, C4, C6, C8, M, O2, P)
Klebsiella pneumoniae (Diagnostic Serial 12073) (A2, A3) 20 16 17 20 3.5
Numbers in brackets indicate resistance to the following antibiotics: Amikacin (A1), Amoxycillin (A2), Ampicillin (A3),
Augmentin (A4), Cefataxamine (C1), Cefataxime (C2), Ceflotaxine (C3), Cefoparazene (C4), Ceforaxin (C5), Ceftazidine
(C6), Ceftriaxone (C7), Cefuroxime (C8), Cephalexin (C9), Ciprofloaxin (C10), Cloxacillin (C11), Cotrimoxazole (C12),
Doxycyclin (D), Gentamicin (G), Kanamycin (K), Methicillin (M), Nalidixic Acid (N1), Netilmicin (N2), Norfloxacin (N3),
Oflaxacin (O1), Oxacillin (O2), Piperacillin (P), Sparfloxacin (S)
a
Diameter of cup was 7 mm. All determinations were performed at least thrice and the average of the values are
reported
b
For purified compound of Actinobacterium MS1/7
c
Did not preserve its drug-resistant property throughout the period of study

123
1086 Biotechnol Lett (2006) 28:1083–1088

range of bacteria and fungi. Our compound

inhibited the growth by 54% of human leukemia
cell line (HL-60) at 0.05 lg ml)1 compared to
200 lg ml)1 required for cytosine arabinoside.

Toxicity

The active compound of the isolate MS1/7 lysed

only 2.3% of murine erythrocytes and 1.6% of
human erythrocytes at concentrations 10 times
the MIC values.
Fig. 1 Predicted structure of the active compound
Characterization of the antimicrobial from Actinobacterium MS1/7: 4a,8a-dimethyl-6-
substance (2-methyl-propenyloxy)-3,4,4a,4b,5,6,8a,9-octahydro-1H-
phenanthre n-2-one
The IR spectrum of the active principle showed
structure of our compound. The other databases
the presence of a six-member carbonyl group
(Novel Antibiotics Database, PubChem, Sci-
at 1738 cm)1 and C–O stretching of dimethyl-
Finder Scholar, SCOPUS, NIST and US, Euro-
vinyl ether (CH3)–CHACH–O– at 1242 cm)1 and
pean and Japanese Patents Database) were
1081 cm)1. High-resolution mass spectrum
searched, but neither any known bioactive sub-
showed a molecular ion peak at m/z 301.2101
stance nor any known toxin was found possessing
[M+H]+ and indicated a molecular formula of
the antagonistic properties described.
C20H28O2 along with peaks at m/z 323.2304 and
339.2208 for [M+Na]+ and [M+K]+ respectively. Production in a bioreactor
The proton NMR spectrum of the compound
exhibited signals for methyl groups of C-4a, C-8a The time course of the production of the antimi-
and for methylene protons of C-4 at d 0.89–0.98 crobial substance during the best operating con-
(m, 8H, 2x-CH3, 1x-CH2). The two methyl groups dition with aeration rate of 0.5 l min)1 and
attached to the vinyl carbon appeared at d1.25– agitation of 200 rpm (minimum pO2 40%
1.36 (m, 7H, 2x-CH3, 1x-CH), including one me-
thine proton of C-4b. The methylene protons of

Zone of inhibition
Residual maltose (gl )
-1
C-2, C-5 and C-9 appeared at d1.56–1.66 (m, 6H, 0.05

(mm)
Growth (as intracellular protein, g l-1)

5
3x-CH2), while the other methylene protons of 30
0.04
C-1 and methine proton of C-6 emerged at d 2.05 8.00 4
25
(t, J = 6.3 Hz, 3H, 1x-CH2, 1x-CH). The olefinic 0.03
7.75 3
protons of C-7, C-8, and C-10 produced signals at 20
pH

0.02 2
d 3.90–4.41 (m, 3H, 3x-CH) and a vinyloxy proton 7.50
15
appeared at d 4.60 (s, 1H). The proposed struc- 0.01
1
7.25 10
ture on the basis of the above evidences is shown 0
in Fig. 1. 0.00 7.00 5
0 20 40 60 80 100 120
Time (h)
Electronic database search
Fig. 2 Time course of the production of the antimicrobial
substance by Actinobacterium MS1/7 in a 2.5 l bioreactor.
ChemIDplus (Advanced) database (containing d residual maltose (g l)1), n growth as intracellular protein
370,000 compounds) was searched with classifi- (g l)1), pH, antimicrobial activity (zone of inhibition, mm,
cation codes ‘‘antibiotic’’, ‘‘anti-cancer’’, ‘‘poi- represented as the average of the two diameters against
S. aureus and A.niger at each sampling time). The diameter of
son’’ and ‘‘hazardous’’ in the molecular weight
the cup for testing antimicrobial activity is 7 mm. The
range 297–303. Eighteen hits were obtained but bioreactor was run twice and the results were reproducible
none matched the properties and molecular within less than 2% deviation

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Biotechnol Lett (2006) 28:1083–1088
Fig. 3 Phylogenetic tree showing the position of the isolate Actinobacterium MS 1/7 in the genus Streptomyces
saturation) is shown in Fig. 2. The synthesis of the
antimicrobial substance is unusually associated
with growth.
Characteristics of the producing
microorganism
The isolate MS1/7 is a Gram-positive spore-
forming bacterium, which produces white aerial
and substrate mycelium. It has no obligate
requirement of NaCl for growth but can tolerate
upto 200 g NaCl l)1 and the biomass increase (up
to 100 g NaCl l)1) displayed a graded response to
variations in NaCl concentration indicating its
intertidal habitat (Mincer et al. 2002). Based on
the sequence of the 16S rRNA gene (GenBank
Accession No., AY550275), isolate MS1/7 belongs
to the genus Streptomyces (Fig. 3); distinct from
other salt-tolerant actinobacteria and closely
related to our earlier isolate, MS3/20 (GenBank
Accession No. AY 144607, Saha et al. 2005).
Maldonado et al. 2005 mentioned that use of in-
formed selective isolation procedures can aid in
the isolation of novel actinobacteria from geo-
graphically widespread marine sediments.
Conclusion
Although RBC hemolysis is a criteria for deter-
mining toxicity of antimicrobials (Saha et al.
2005) and anticancer agents (Jendrossek et al.
2002; Taylor et al. 1989), a clear picture of the
toxic effects will emerge only after animal studies
are completed. We believe that our screening
1087
strategy should increase the possibility of discov-
ering bioactive molecules from rare actinobacte-
ria (Gathogo et al. 2004). The isolation of the
marine actinobacterium is a significant finding as
it is the first marine bacterial source producing a
single compound active against MDR bacteria
and leukemia cells. Previously, a marine sponge,
Arenosclera brasiliensis, was found to produce
compounds with similar inhibitory properties
(Torres et al. 2002).
Acknowledgements Financial support from the AICTE
[Grant No. 8019/RDII/R&D/BIO(135)2000-01], DST
(Grant No. SR/FTP/LS-A-016/2001) to J. Mukherjee,
ICMR fellowships to M. Saha (No. 45/26/2001/Pharma/
BMS) and B. Sana (No. 45/1/2004/Pharma/BMS) is
thankfully acknowledged. Suggestions of Dr. Tuhinadri
Sen, Jadavpur University are highly appreciated.
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