ORIGINAL PAPER

Cyclic dipeptides from rhabditid entomopathogenic nematode-associated Bacillus cereus have antimicrobial activities

S. Nishanth Kumar • Vishnu Sukumari Nath •

R. Pratap Chandran • Bala Nambisan

Received: 17 May 2013 / Accepted: 10 August 2013 / Published online: 24 August 2013

� Springer Science+Business Media Dordrecht 2013

Abstract The cell free culture filtrate of Bacillus cereus

associated with an entomopathogenic nematode, Rhabditis

(Oscheius) sp. exhibited strong antimicrobial activity. The

ethyl acetate extract of the bacterial culture filtrate was

purified by silica gel column chromatography to obtain

four bioactive compounds. The structure and absolute

stereochemistry of these compounds were determined

based on extensive spectroscopic analyses (FABMS, 1H

NMR, 13C NMR, 1H–1H COSY, 1H–13C HMBC) and

Marfey’s method. The compounds were identified as cyclic

dipeptides (CDPs): cyclo(L-Pro-L-Trp), cyclo(L-Leu-L-Val),

cyclo(D-Pro-D-Met), and cyclo(D-Pro-D-Phe), respectively.

Compounds recorded significant antibacterial activity

against all the test bacteria (Staphylococcus epidermidis,

Staphylococcus aureus, Klebsiella pneumoniae, Esche-

richia coli, Pseudomonas aeruginosa and methicillin-

resistant S. aureus) except cyclo(L-Leu-L-Val). Cyclo(L-

Leu-L-Val) recorded activity only against Gram positive

bacteria. Best antibacterial activity was recorded by

cyclo(L-Pro-L-Trp) against S. aureus (4 lg/ml). The four

compounds were active against all the five fungi tested

(Trichophyton rubrum, Aspergillus flavus, Candida albi-

cans, Candida tropicalis and Cryptococcus neoformans)

and the activity was compared with amphotericin B, the

standard fungicide. The highest activity of 1 lg/ml by

cyclo(L-Pro-L-Trp) was recorded against T. rubrum, a

human pathogen responsible for causing athlete’s foot, jock

itch, and ringworm. The activity of cyclo(L-Pro-L-Trp)

against T. rubrum, C. neoformans and C. albicans were

better than amphotericin B, the standard antifungal agent.

To our knowledge, this is the first report of antifungal

activity of CDPs against the human pathogenic fungi T.

rubrum and C. neoformans. The four CDPs are nontoxic to

healthy human cell line up to 200 lg/ml. We conclude that

the bacterium associated with entomopathogenic nematode

is promising sources of natural antimicrobial secondary

metabolites, which may receive greater benefit as potential

sources of new drugs in the pharmaceutical industry.

Keywords Bacillus cereus � Secondary metabolite �Purification � Pharmaceutical

Introduction

The entomopathogenic nematode/bacterium (EPN/EPB)

symbiotic associations are considered model systems to

address broad biological questions of mutualism, co-evo-

lution and pathogenesis (Boszormenyi1 et al. 2009). The

bacterial genera Photorhabdus and Xenorhabdus are phy-

logenetic sister groups belonging to the family Entero-

bacteriaceae; they are associated with entomopathogenic

nematodes of the genera Heterorhabditis and Steinernema,

respectively (Tailliez et al. 2010). During the symbiotic

stage, the bacteria are carried in the nematode gut, but after

infection of an insect host, the nematodes inject the

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11274-013-1461-7) contains supplementarymaterial, which is available to authorized users.

S. Nishanth Kumar � V. S. Nath � B. Nambisan (&)

Division of Crop Protection/Division of Crop Utilization,

Central Tuber Crops Research Institute, Sreekariyam,

Thiruvananthapuram 695017, India

e-mail: balactcri@gmail.com

R. Pratap Chandran

Department of Biotechnology and Research, K. V. M. College of

Engineering and Information Technology, K. V. M. College

Road, Kokkothamangalam P. O., Cherthala,

Alappuzha District 688583, Kerala, India

123

World J Microbiol Biotechnol (2014) 30:439–449

DOI 10.1007/s11274-013-1461-7

bacteria into the insect hemocoel (Forst et al. 1997). The

bacteria multiply rapidly and produce various metabolites,

which can overcome the insect immune system (Forst and

Nealson 1996), kill the insect, and inhibit the growth of

various fungal and bacterial competitors (Akhurst 1982;

Chen et al. 1994, 1996). By doing so, the bacterial sym-

bionts are believed to prevent putrefaction of the insect

cadaver and establish conditions that favour the develop-

ment of both the nematode and bacterial symbionts (Gau-

gler and Kaya 1990).

The antimicrobial nature of metabolites produced by

Xenorhabdus spp. and Photorhabdus spp. is known, and

several compounds with biological activity have been iso-

lated and identified. These include indoles and stilbenes

(Paul et al. 1981; Li et al. 1995), xenorhabdins (McInerney

et al. 1991a), xenocoumacin (McInerney et al. 1991b), ne-

matophin (Li et al. 1997), benzylineacetone (Ji et al. 2004),

xenortides and xenematide (Lang et al. 2008), and cyclo-

lipopeptide (Gualtieri et al. 2009). An area with ramifica-

tions in plant pathology, veterinary science, and even human

health, is the secondary metabolites produced by EPBs.

In the course of studies on EPN, a new entomopatho-

genic nematode belonging to the genus Rhabditis and

subgenus Oscheius was isolated from sweet potato weevil

grubs collected from Central Tuber Crops Research Insti-

tute (CTCRI) farm, Thiruvananthapuram. A specific bac-

terium was found associated with the nematodes. The

nematodes could be cultured on laboratory reared Galleria

mellonella larvae and maintained alive for several years.

The bacteria were found to be pathogenic to a number of

insect pests (Mohandas et al. 2007) and could be isolated

from 3rd stage infective juveniles of the nematode or from

the hemolymph of nematode infested G. mellonella larvae.

Based on molecular characteristics, Rhabditis (Oscheius)

sp. resembles Rhabditis isolate Tumian 2007 at D2 and D3

(nucleotide sequence region) expansion segments of 28S

rDNA (Deepa et al. 2010). The cell free culture filtrate of

the bacteria was found to inhibit several pathogenic bac-

teria, fungi and a plant parasitic nematode (Meloidogyne

incognita) (Mohandas et al. 2007), suggesting that it could

be a rich source of biologically active compounds. In this

paper, we report the isolation, structure elucidation and

antimicrobial activity of the bioactive secondary metabo-

lites from the cell free culture filtrate of the bacterium with

special references to human pathogenic fungi.

Materials and methods

Chemicals and media

All the chemicals used for extraction and column chro-

matography were of analytical grade. High performance

liquid chromatography (HPLC) grade methanol was from

Merck Ltd., Mumbai, India. Silica gel (230–400 mesh)

used for column chromatography and precoated silica gel

60 GF254 plates used for Thin Layer Chromatography

(TLC) were from Merck Ltd., Germany. Microbiological

media were from Hi-Media Laboratories Ltd., Mumbai,

India. The standard antibiotics ciprofloxacin and ampho-

tericin B were purchased from Sigma Aldrich. The soft-

ware used for the chemical structure drawing was

Chemsketch Ultra, Toranto, Canada.

Test microorganisms

Bacteria and fungi as follows were used in the present

study. Gram positive bacteria: Staphylococcus epidermidis

MTCC 10623, Staphylococcus aureus MTCC 902; Gram

negative bacteria: Klebsiella pneumoniae MTCC 109,

Escherichia coli MTCC 2622, and Pseudomonas aerugin-

osa MTCC 2642; medically important fungi: Trichophyton

rubrum MTCC 296, Aspergillus flavus MTCC 183, Can-

dida albicans MTCC 277 Candida tropicalis MTCC 184.

All the test microorganisms were purchased from Micro-

bial Type Culture collection Centre, IMTECH, Chandi-

garh, India. Methicillin-resistant S. aureus (MRSA) was

obtained from the Department of Medical Microbiology,

Government medical college hospital, Trivandrum.

Isolation of Bacillus cereus

The bacterial strain N was isolated from 3rd stage infective

juveniles of the nematode sample collected from sweet

potato weevil grubs or from the hemolymph of nematode

infested G. mellonella larvae. The strain was identified as

Bacillus cereus (Accession No. HQ200404) based on 16S

rDNA and BLAST analysis. The strain was currently

deposited in IMTECH (Institute of Microbial Technology,

Chandigarh; India) and the accession number is MTCC

5234.

Incubation and extraction

The bacterial incubation was carried out using modified

nutrient broth (NB) (peptic digest of animal tissue 5 g/l,

NaCl 5 g/l, yeast extract 1.5 g/l, beef extract 1.5 g/l, water

1,000 ml) supplemented by glucose 5 g/l and 0.1 % tryp-

tophan. A single colony of Bacillus sp. from the agar plate

was inoculated into the flask containing 100 ml sterile

media. The flasks were incubated in a gyrorotatory shaker

(150 rpm) at 30 �C in dark for 24 h. When the optical

density of the culture at 600 nm was approx 1.7, the bac-

terial cultures were transferred aseptically into 400 ml

sterile medium and incubated in the gyrorotatory shaker at

30 �C in dark for 96 h. The culture media were then

440 World J Microbiol Biotechnol (2014) 30:439–449

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centrifuged (10,000g, 20 min, 4 �C) followed by filtration

through a 0.45 lm filter, to obtain cell free culture filtrate.

Thirty litres of cell free culture filtrate were neutralized

with concentrated hydrochloric acid and extracted with an

equal volume of ethyl acetate thrice. The ethyl acetate

layers were combined, dried over anhydrous sodium sul-

phate, and concentrated at 30 �C using a rotary flash

evaporator.

Purification of bioactive compounds

The oily yellow residue (9.3 g) obtained after drying was

then loaded on a silica gel column (25 9 600 mm) previ-

ously equilibrated with hexane and eluted successively with

200 ml of 100 % hexane, 200 ml of linear gradient hexane:

dichloromethane (v/v, 75:25–25:75), 200 ml of 100 %

dichloromethane, 200 ml of linear gradient dichlorometh-

ane:ethyl acetate (v/v, 95:5–5:95), 200 ml of 100 % ethyl

acetate and finally with 200 ml of 100 % methanol. Two

fractions (100 ml each) were collected from each combi-

nation. Four fractions yielded white crystal compounds,

which were further purified by crystallization using hexane

and benzene. The antibacterial activity of these fractions

was determined by well diffusion assay against B. subtilis,

which was selected as initial test microorganism.

The purity of the compounds was checked using TLC

(silica gel) and HPLC, using LC-10AT liquid chromatography

(LC; Shimadzu, Singapore) equipped with a C-18 column

(5 lm, 4.6 9 250 mm) and 100 % methanol as a mobile

phase with a flow rate of 1 ml/min. Ultraviolet (UV) detection

was carried out with a diode array detector (Shimadzu).

Spectroscopic measurements

UV spectrophotometer

UV–visible spectrum of the pure compounds was recorded

on a Systronics double beam spectrophotometer 2201,

India at room temperature (scanning range 190–800 nm).

FABMASS

FABMASS was performed on a JEOL JMS-SX/SX102A

four-sector tandem MS (JEOL, Ltd., Tokyo, Japan) with a

fast-atom-bombardment (FAB) ion source with glycerol as

the matrix.

Nuclear magnetic resonance (NMR)

The structure of the compounds was determined using nuclear

magnetic resonance (NMR) spectroscopy (Bruker DRX 500

NMR instrument, Bruker, Rheinstetten, Germany) equipped

with a 2.5-mm microprobe. NMR Spectrometer using CDCl3

was deployed to measure 1H and 13C and 2D NMR. All spectra

were recorded at 23 �C. One-dimensional 1H NMR experi-

ments as well as two-dimensional 1H–1H correlation spec-

troscopy, 1H–13C heteronuclear multiple bond correlation,

and 1H–13C heteronuclear multiple quantum coherence

(HMQC) experiments were performed according to Bruker

standard pulse sequences. Proton chemical shifts were deter-

mined from one-dimensional 1H NMR and from HMQC

experiments, and 13C chemical shifts were determined from

HMQC and 1H–13C heteronuclear multiple bond correlation

experiments. Chemical shifts are reported relative to the sol-

vent peaks. (CDCl3: 1H d 7.24 and 13C d 77.23).

Optical rotations

Optical rotation of the compounds was measured using a

Rudolph Research Autopol III polarimeter at 25 �C in

acetone.

Differential scanning calorimetry

The melting point of the pure compounds was measured with a

differential scanning calorimeter with a Mettler Toledo DSC

822e instrument (Mettler-Toledo, Schcoerfenbach, Switzer-

land). Temperature ranges from 30 to 300 �C were employed.

Absolute configuration determination of compounds

by Marfey’s method

A solution of three compounds (1.5 mg) in 6 M HCl (1 ml)

was heated to 120 �C for 24 h. The solution was then

evaporated to dryness and the residue redissolved in H2O

(100 ll) and was then placed in a 1 ml reaction vial and

treated with a 2 % solution of FDAA (200 ll) in acetone

followed by 1.0 M NaHCO3 (40 ll). The reaction mixture

was heated at 47 �C for 1 h, cooled to room temperature,

and then acidified with 2.0 M HCl (20 ll). In a similar

fashion, standard D- and L-amino acids were derivatized

separately. The derivatives of the hydrolysates and stan-

dard amino acids were subjected to HPLC analysis (Shi-

madzu LC-20AD, C18 column; 5 lm, 4.6 9 250 mm;

1.0 ml/min) at 30 �C using the following gradient program:

solvent A, water ? 0.2 % TFA; solvent B, MeCN; linear

gradient 0 min 25 % B, 40 min 60 % B, 45 min 100 % B;

UV detection at 340 nm (Marfey 1984).

Determination of antibacterial activity

Minimum inhibitory concentration (MIC)

Minimum inhibitory concentration was determined by

standard macro dilution broth test as recommended by the

National Committee for Clinical Laboratory Standards, USA

World J Microbiol Biotechnol (2014) 30:439–449 441

123

(CLSI 2006) against all the four test bacteria. The pure

compounds and standard antibiotics were tested at final

concentrations, prepared from serial twofold dilutions,

ranging from 1 to 2,000 lg/ml. The MIC was defined as the

lowest concentration of the test compound that prevented

visible growth of test bacteria. Triplicate sets of tubes were

maintained for each concentration of the test sample.

Minimum bactericidal concentration (MBC)

Minimum bactericidal concentration was determined

according to the method of Smith-Palmer et al. (1998)

against all the four test bacteria. About 100 ll culture fil-

trate from the tubes showing no growth in the MIC test

were plated on nutrient agar. MBC is the lowest concen-

tration of test compound at which bacteria failed to grow in

nutrient broth and nutrient agar inoculated with 100 ll of

suspension. Triplicate sets of tubes were maintained for

each concentration of the test sample.

Determination of antifungal activity

Minimum inhibitory concentration

Minimum inhibitory concentration was determined using

potato dextrose agar media against the standard fungicide

bavistin by the poisoned food technique (Rollas et al. 1993)

against except Candida spp. A stock solution of 1,000 lg/

ml of the test compound was prepared, which was further

diluted with methanol to give the required concentrations

1,000–1 lg/ml. One tube was used as solvent control. For

C. albicans and C. tropicalis, the broth dilution method

was adopted using potato dextrose broth against the stan-

dard fungicide amphotericin B. All experiments were in

triplicate for each treatment against each fungus.

Agar disc diffusion method

In vitro antibacterial and antifungal activity of the com-

pounds was measured using an agar disc diffusion assay

against the test bacteria and fungi (Murray et al. 1995;

CLSI 2008). The sterile disks were impregnated with MIC

concentration of test compounds. The ciprofloxacin was

used as positive reference standards for bacteria. Ampho-

tericin B was used as reference standard for fungus. The

antimicrobial activity was evaluated by measuring the zone

of growth inhibition surrounding the disks. All the assays

were carried out in triplicate.

Cytotoxicity test

The MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tet-

razolium bromide) assay was used to determine the

cytotoxicity of compounds. FS normal fibroblast cell line

was used for testing. MTT assay is based on the ability of

mitochondrial dehydrogenase enzyme from viable cells to

cleave the tetrazolium rings of the pale yellow MTT and to

form dark blue formazan crystals, which are largely

impermeable to cell membranes, thus resulting in its

accumulation within healthy cells. Solubilization of the

cells by the addition of a detergent results in the liberation

of the crystals. The number of surviving cells is directly

proportional to the level of the formazan product formed.

The colour can then be quantified by a simple colorimetric

assay using a multi-well scanning spectrophotometer

(ELISA reader). Briefly, cells (5 9 103/well) were seeded

in 0.2 ml of the medium (DMEM with 10 % PBS) in 96

well plates, treated with drugs for 72 h and after incuba-

tion, cytotoxicity was measured. For this after removing

the drug containing media, 25 ll of MTT solution (5 mg/

ml in PBS) and 75 ll of complete medium were added to

wells (untreated and treated) and incubated for 2 h. At the

end of incubation MTT lysis buffer was added to the wells

(0.1 ml/well) and incubated for another 4 h at 37 �C. At

the end of incubation, the optical densities at 570 nm were

measured using a plate reader (Biorad ELISA reader 680,

California, USA). The relative cell viability in percentage

was calculated (A570 of treated sample/A570 of untreated

sample 9 100) (Anto et al. 2003).

Statistical analysis

Statistical analyses were performed with SPSS (Version

17.0; SPSS, Inc., Chicago, IL, USA). Data for disc diffu-

sion assay was presented as means ± standard deviations.

Statistical significance was defined as p \ 0.05.

Results

Isolation and purification of bioactive compounds

The ethyl acetate extract of the cell free culture filtrate of

the bacteria showed antibacterial activity against B. sub-

tilis. Silica gel column chromatography of this extract

yielded four crystal compounds. The column solvent and

yield were shown in the Table 1. These crystal compounds

were further purified by crystallization using hexane and

benzene. Initial bioactivity of these compounds was con-

firmed by testing against the indicator test microorganism

B. subtilis. Thin layer chromatography of the purified

compounds revealed single spots and RF value is presented

in the Table 1. HPLC analysis of the four compounds was

performed by reverse phase and compounds were eluted as

single peaks (Table 1). The purity of the compounds

reached greater than 90 % according to the peak area.

442 World J Microbiol Biotechnol (2014) 30:439–449

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Identification of bioactive compound

The pure compounds were subjected to various spectro-

scopic analyses, i.e. UV, FABMS and NMR. The structure

of these four compounds corresponded to four different

diketopiperazines or cyclic dipeptides (CDPs). The com-

pounds identified are cyclo(L-Pro-L-Trp), cyclo(L-Leu-L-

Val), cyclo(D-Pro-D-Met), and cyclo(D-Pro-D-Phe), respec-

tively (Fig. 1).

CDP 1: Cyclo(L-Pro-L-Trp) (3S,8aS)-3-(1H-indol-3-

ylmethyl)hexahydropyrrolo[1,2-a]pyrazine-1,4-dione: was

obtained as white crystalline powder; Melting point:

285.58 �C (Fig. 2A); [a]D-128 (c, 0.02, MeOH); UV max:

220 nm (MeOH); NMR data (supplementary data); Based

on the spectral data the molecular formula was determined

to be C16H17N3O2 by FABMS [m/z 284.34 (M ? H)],

calcd. for C16H17N3O2, 283.32.

CDP 2: Cyclo(L-Leu-L-Val) (3S,6S)-3-(2-methylpropyl)-

6-(propan-2-yl)piperazine-2,5-dione: was obtained as solid

powder; Melting point: 267.71 �C (Fig. 2B); [a]D-91 (c,

0.02, MeOH); UV max: 215 nm (MeOH); NMR data

(supplementary data); Based on the spectral data the

molecular formula was determined to be C11H20N2O2 by

FABMS [m/z 213.84 (M ? H)], calcd. for C11H20N2O2,

212.28.

CDP 3: Cyclo(D-Pro-D-Met) (3R,8aR)-3-[2-(methylsulfa-

nyl)ethyl]hexahydropyrrolo[1,2-a]pyrazine-1,4-dione: was

obtained as white amorphous powder; Melting point:

181.60 �C (Fig. 2C); [a]D30 ?85.3� (c 0.10, EtOH); UV max:

210 nm (MeOH); NMR data (supplementary data); Based on

the spectral data the molecular formula was determined to be

C10H17O2N2S by FABMS [m/z 229.10 (M ? H)] calcd. for

m/z 228.13.

CDP 4: Cyclo(D-Pro-D-Phe) (3R,8aR)-3-benzylhexahy-

dropyrrolo[1,2-a]pyrazine-1,4-dione: was obtained as

white amorphous powder; Melting point: 156.28 �C

(Fig. 2D); [a]D30 ?76.91� (c 0.04, EtOH); UV max: 208 nm

(MeOH); NMR data (supplementary data); Based on the

spectral data the molecular formula was determined to be

C10H17O2N2S by FABMS [m/z 245.12 (M ? H)] calcd. for

m/z 244.19.

Absolute configuration determination of CDPs

The modified Marfey’s method was successfully applied to

the determination of the absolute configuration of com-

pounds. Derivatives obtained from the hydrolysis of the

CDPs were compared with the retention times of the

derivatized standard D- and L-amino acids. The retention

times for the FDAA derivatives of the four CDPs and

corresponding standard amino acids are presented in the

Fig. 3. Regarding the absolute stereochemistry, the CDP 1

and 2 contains both L-amino acid (Fig. 3A, B) where as

CDP 3 and 4 contains both D-amino acids (Fig. 3C, D).

Bioactivity

Antibacterial activity

The isolated CDPs were tested for antibacterial activity

against test bacterial strains using standard methods. MIC

and MBC values were determined and are shown in

Table 2. The microorganism that presented highest sensi-

tivity towards CDP 1 was S. aureus (4 lg/ml), followed by

S. epidermidis (8 lg/ml) and P. aeruginosa (16 lg/ml).

Table 1 Isolation and purification details of pure compounds

Compound Column solvent Yield

(mg)

RF

value

Retention

time

(min)

1 20 % ethyl acetate in DCM 15 0.52 3.459

2 27 % ethyl acetate in DCM 13 0.44 2.944

3 42 % ethyl acetate in DCM 9 0.23 2.735

4 68 % ethyl acetate in DCM 17 0.36 2.619

Fig. 1 Structure of CDPs. A Cyclo(L-Pro-L-Trp), B cyclo(L-Leu-L-

Val), C cyclo-(D-Pro-D-Met), D cyclo-(D-Pro-D-Phe)

World J Microbiol Biotechnol (2014) 30:439–449 443

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CDP 2 was active only against Gram positive bacteria and

the best activity of this CDP was recorded against S. aureus

(32 lg/ml). CDP 3 presented highest activity S. epide-

rmidis (16 lg/ml). CDP 4 recorded highest activity against

E. coli (16 lg/ml). It appeared that effective MIC also

represents the effective bactericidal concentration of the

bacteria tested. The activity of the test CDPs was lower

than the standard antibiotic ciprofloxacin. Except CDP 1

(16 lg/ml) all other CDPs were not active against MR—S.

aureus.

Antifungal activity

Antifungal activity against five fungi and corresponding

MIC values are indicated in Table 3. Four CDPs exhibited

good antifungal activity against all the tested fungi espe-

cially against T. rubrum, C. albicans, C. tropicalis and C.

neoformans. CDP 1 recorded highest activity against T.

rubrum (1 lg/ml) followed by C. neoformans (2 lg/ml).

CDPs recorded significant activity against human patho-

genic fungi. The antifungal activity of CDPs against C.

tropicalis and C. albicans in comparison with amphotericin

B, are in Table 2. CDP 1 recorded activities in 4 and 8 lg/

ml, respectively for C. albicans and C. tropicalis, where as

amphotericin B recorded activity at 2 and 16 lg/ml. The

antifungal activity of the CDPs was comparable with the

activity of the standard fungicide amphotericin B against

all the five fungi tested (Table 3).

Agar disc diffusion assay

The result of the disc diffusion assay against the test

microorganisms is presented in the Table 4. CDP 1

recorded best antimicrobial activity against test bacteria.

CDP 1 recorded highest antibacterial activity against

E. coli and S. aureus (28 mm) followed by S. epidermidis

and P. aeruginosa (26 mm) (Table 4; Fig. 4). The best

antifungal activity of CDP 1 was recorded against T. ru-

brum (33 mm) followed by C. neoformans (32 mm). CDP

2 and 3 also recorded best activity against T. rubrum (32

and 24 mm, respectively). Whereas CDP 4 recorded best

activity against C. neoformans (31 mm).

Cytotoxicity test

Cytotoxicity activity of CDPs was determined by MTT

assay after 72 h of treatment. The four CDPs are nontoxic

to healthy human cell line up to 200 lg/ml (Fig. 5). At

200 lg/ml of CDPs, more than 85 % of the cells were alive

(Fig. 5). This clearly indicated that CDPs are safe for

Fig. 2 DSC curves of CDPs. Temperatures corresponding to the onset of transition and midpoint of the transition region and enthalpy (DH) were

recorded by means of the built-in software. A Cyclo(L-Pro-L-Trp), B cyclo(L-Leu-L-Val), C cyclo-(D-Pro-D-Met), D cyclo-(D-Pro-D-Phe)

444 World J Microbiol Biotechnol (2014) 30:439–449

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therapeutic purposes and its action may be selectively

targeted against the bacteria and fungi.

Discussion

The 2,5-diketopiperazines (CDPs), head-to-tail dipeptide

dimers, are a common naturally occurring skeleton (Prasad

1995). Diketopiperazines corresponding to cyclic dipep-

tides have been isolated from microorganisms, sponges and

from a variety of tissues and body fluids (Rudi et al. 1994;

Strom et al. 2002; De Rosa et al. 2003). Due to their rel-

ative simplicity (Anteunis 1978) and stability (Prasad

1995), diketopiperazines provide excellent models for

theoretical studies as well as the development of pharma-

ceutical compounds. Diketopiperazines possess diverse

Fig. 3 HPLC profile of FDAA derivatives of the acid hydrolysates of CDPs. A Cyclo(L-Pro-L-Trp), B cyclo(L-Leu-L-Val), C cyclo-(D-Pro-D-

Met), D cyclo-(D-Pro-D-Phe)

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biological activities such as antitumor (Nicholson et al.

2006; van der Merwe et al. 2008), antifungal (Houston

et al. 2004), antibacterial (Fdhila et al. 2003), and antihy-

perglycemic (Song et al. 2003) activities. Due to their

chiral, rigid, and functionalized structures, they bind to a

large variety of receptors with high affinity, giving a broad

range of biological activities (Martins and Carvalho 2007).

Therefore, diketopiperazines are attractive structures for

the discovery of new lead compounds for the rational

development of new therapeutic agents. In the present

study, we have isolated four cyclic dipeptides (CDPs 1–4)

from ethyl acetate extract of the cell free culture filtrate of

Bacillus sp. associated with rhabitid entomopathogenic

nematode.

Table 2 MIC and MBC (lg/ml) of CDPs against bacteria

Test compound S. epidermidis S. aureus E. coli P. aeruginosa K. pneumoniae MR— S. aureus

MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC

CDP 1 8 16 4 8 32 32 16 16 64 64 16 32

CDP 2 128 128 32 64 – – – – – – – –

CDP 3 16 16 32 64 250 500 500 500 250 500 – –

CDP 4 64 128 64 64 16 32 128 128 125 125 1000 –

Ciprofloxacin 4 4 2 4 2 2 4 8 1 1 4 8

Values represent mean of three replications

– no MIC up to 1,000 lg/ml

Table 3 MIC of CDPs against fungi

Test compound MIC (lg/ml)

A. flavus C. albicans C. tropicalis T. rubrum C. neoformans

CDP 1 32 8 4 1 2

CDP 2 250 32 16 32 16

CDP 3 64 64 16 8 32

CDP 4 64 128 32 8 16

Amphotericin B 32 16 2 2 4

Values represents mean of three replications

Table 4 Antimicrobial activity of CDPs

Test organism Zone of inhibition (dia. in mm)

CDP 1 CDP 2 CDP 3 CDP 4 Ciprofloxacin Amphotericin B

A. flavus 23 ± 1b 20 ± 0b 15 ± 0b 21 ± 0b * 24 ± 1.52c

C. albicans 17 ± 1a 18 ± 0.57a 18 ± 0.57c 16 ± 0.57a * 24 ± 1.73c

C. tropicalis 22 ± 1.52b 21 ± 0b 20 ± 1.15d 30 ± 0e * 16 ± 0a

T. rubrum 33 ± 1d 31 ± 0.57d 24 ± 1.15e 28 ± 0.57d * 19 ± 1.52b

C. neoformans 32 ± 0.57d 29 ± 0.57c 23 ± 1.15e 31 ± 0.57e * 24 ± 1.15c

S. epidermidis 26 ± 1b 18 ± 0a 13 ± 1a 23 ± 0c 31 ± 0.57d *

S. aureus 28 ± 0c 20 ± 0.57b 15 ± 1.73b 20 ± 0.57a 31 ± 0d *

E. coli 28 ± 0.57c – 15 ± 0b 25 ± 0.57 28 ± 1.52c *

P. aeruginosa 26 ± 0b – 17 ± 1.15c 22 ± 0b 25 ± 0.57b *

K. pneumoniae 23 ± 0.57b – 19 ± 1d 15 ± 2.12a 27 ± 1.15c *

MR—S. aureus 20 ± 0.57a – 17 ± 0.57c 16 ± 0.57a 23 ± 1.52a *

Values followed by different letters in same column were significantly different according to Duncan’s multiple range test p = 0.05

– not tested as the MIC value is above 100 (lg/ml), * not tested

446 World J Microbiol Biotechnol (2014) 30:439–449

123

The isolation of cyclo(Pro-Trp) CDP 1 was previously

reported from Sulfitobacter sp., deep-sea bacterium Strep-

tomyces fungicidicus and Streptomyces sp. H7372 (Long

et al. 2011; Li et al. 2006; Cheenpracha et al. 2011). The

antibacterial activity of this CDP was reported against

E. coli, P. aeruginosa, K. pneumoniae, S. aureus, Bacillus

subtilis, Streptococcus pneumoniae, C. albicans, Aspergillus

niger and Penicillium notatum (Graz et al. 1999) with little

information about the inhibitory studies and we observed

greater potency of this compound against human pathogenic

microorganism and best activity was recorded against T.

rubrum with an MIC value of 1 lg/ml The antilarval activity

of cyclo(Pro-Trp) using the barnacle Balanus amphitrite was

also reported (Li et al. 2006). The maturation of the gastro-

intestinal cells by cyclo(Pro-Trp) was also reported by Graz

et al. (1999). The production of cyclo(Leu-Val) CDP 2 was

previously reported from deep-sea bacterium Streptomyces

fungicidicus (Li et al. 2006) and to our best knowledge there

was no report on the antimicrobial activity of this CDP. Our

results showed that CDP 2 having the good inhibitory

potential against both bacteria and fungi with a remarkable

display of activity against C. neoformans (8 lg/ml).

Cyclo(L-Pro-L-Phe) CDP 3 was previously reported

from Antarctic sponge-associated bacterium, P. aeruginosa

(Jayatilake et al. 1996), Streptomyces fungicidicus (Li et al.

2006), marine bacteria Bacillus subtilis sp. 132 (Wang

et al. 2010). Various biological activities of cyclo(Pro-Phe)

including antifungal (Wang et al. 1999; Strom et al. 2002),

antimicrobial (Graz et al. 1999; Rhee 2006) and quorum

sensing (Holden et al. 1999; Degrassi et al. 2002) proper-

ties have been reported previously. Rhee (2004) also

reported the antibacterial activity of cyclo(Pro-Phe) against

various gram positive and negative bacteria and the data is

almost agreeable with our results. CDP 3 was less explored

for its inhibitory potential against human pathogens bac-

teria and fungi, for e.g., CDP 3 isolated from the symbiont

of the fungus-growing ant Cyphomyrmex minutus (Wang

et al. 1999) have shown weak antifungal activity in the

zone of inhibition studies against C. albicans. Our results

showed cyclo(D-Phe-D-Pro) having good inhibitory poten-

tial against both bacteria and fungi with a remarkable

display of activity against R. solani (8 lg/ml). CDP 3 has

unnatural D-proline and D-phenylalanine amino acid, indi-

cating the importance of chirality of amino acids having an

Fig. 4 Antibacterial activity by

disk diffusion method for

cyclo(L-Pro-L-Trp). 1 Solvent

control, 2 cyclo(L-Pro-L-Trp),

3 ciprofloxacin

Fig. 5 Cytotoxicity of CDPs against FS normal fibroblast cell lines

World J Microbiol Biotechnol (2014) 30:439–449 447

123

effect on the topography of the three-dimensional structure

resulting in increased inhibition.

Cyclo-(L-Pro-L-Met) was first reported from Antarctic

sponge-associated bacterium, P. aeruginosa (Jayatilake

et al. 1996) and marine-derived actinomycete, Nocardiopsis

sp. 03N67 (Shin et al. 2010). Cyclo(L-Pro-L-Met) isolated

from P. aeruginosa was shown to have antimicrobial activity

against B. subtilis, S. aureus, and Micrococcus luteus (Jay-

atilake et al. 1996) with little information about the inhibi-

tory studies. We have observed that cyclo(D-Pro-D-Met) was

potent against T. rubrum and C. neoformans with an MIC of 8

and 16 lg/ml, respectively. The enhanced activity of

cyclo(D-Pro-D-Met) is due to the presence of unnatural D-

amino acids in the CDP, indicating that the stereochemistry

of the CDP play an important role in increased inhibition.

In the present manuscript, we report the antimicrobial

effects of the CDPs assayed against both medicinally

important bacterium and fungi cf., S. epidermidis, S. aureus,

K. pneumonia, E. coli, P. aeruginosa and methicillin-resis-

tant S. aureus, T. rubrum, A. flavus, C. albicans, C. tropicalis

and C. neoformans. The results showed that CDPs exhibit

potent antimicrobial activity especially against medically

important fungi. The human pathogenic fungi T. rubrum

(causes athlete’s foot, jock itch and ringworm), C. neofor-

mans (causes cryptococcosis) and C. albicans (oral thrush

and vaginal infection) were strongly inhibited by CDP 1. The

activity of CDP 1 is better than the standard antifungal agent

amphotericin B. Isolation of cyclo(D-Pro-D-Met), and

cyclo(D-Pro-D-Phe) from Rhabditis EPN bacterial strain

Bacillus sp. is a new finding in literature with complete

structural characterization. In addition, antifungal activity of

these CDPs against human pathogenic fungi T. rubrum and

C. neoformans, is reported here for the first time. The data

presented in this paper clearly indicated that four CDPs

exhibit potent inhibitory against pathogenic fungi in the

range of 1–250 lg/ml.

The origin of CDPs has been questioned, once several

CDPs have been found in fermentation broths and cultures of

yeast, as well as in lichens and fungi (Prasad 1995). It is known

that CDPs can be generated via non-enzymatic cyclization of

linear dipeptides at extremes of temperature (Holden et al.

1999). It was checked whether CDPs would have been gen-

erated by heat sterilization and incubation of the media culture

during the fermentation process. However, these CDPs were

not detected in the HPLC profiles of the obtained extracts from

the culture medium without the bacterium.

Conclusion

EPN bacteria produce a diverse group of secondary metabo-

lites, of which a few have been isolated and identified. Further,

there are also a number of EPN bacteria that have not been

exploited for their bioactive metabolites. The results of the

present study show that CDPs exhibit strong effects against

test microorganisms especially against medically important

fungi in impressive low concentrations. Therefore, these

microbial secondary metabolites can be ideal candidates for

use as potential antimicrobial agents, and entomopathogenic

bacteria can be regarded as a novel source of potential phar-

maceuticals. Thus, CDPs isolated from bacteria are an

encouraging bioprobe to develop new antifungal therapeutics

from such type of small molecules in the near future.

Acknowledgments The authors are grateful to Indian Council

Medical Research (ICMR), Government of India for funding. We

thank the Director, CTCRI, for providing facilities for the work.

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