Microbiological Research 207 (2018) 116–133

Contents lists available at ScienceDirect

Microbiological Research
journal homepage: www.elsevier.com/locate/micres

Desert actinobacteria as a source of bioactive compounds production with a T

special emphases on Pyridine-2,5-diacetamide a new pyridine alkaloid
produced by Streptomyces sp. DA3-7
Krishnasamy Nithyaa, Chinnasamy Muthukumarb, Bhaskar Biswasc, Naiyf S. Alharbid,
⁎
Shine Kadaikunnand, Jamal M. Khaledd, Dharumadurai Dhanasekarana,e,
a
Department of Microbiology, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India
b
Department of Botany, National College (Autonomous), Tiruchirappalli 620001, Tamil Nadu, India
c
Department of Chemistry, Surendranath College, Kolkata 700009, India
d
Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
e
Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, NH, 03824-2617, USA

A R T I C L E I N F O A B S T R A C T

Keywords: In the present study, 134 morphologically distinct actinobacteria isolates were obtained from soil samples from
Streptomyces sp. 10 different localities in the Saudi Arabian desert. The preliminary screening revealed that 16 of these isolates
Desert soil possessed antimicrobial activity. One isolate, which was identified as Streptomyces sp. DA3-7, possessed broad-
Actinobacteria spectrum antimicrobial activity against both gram-positive and gram-negative bacteria, as well as against fungi,
Cytotoxicity
and modified nutrient glucose medium was suitable for Streptomyces sp. DA3-7 to produce extracellular meta-
Pyridine-2,5-diacetamide
Minimum inhibitory concentration
bolites. The ethyl acetate extract of Streptomyces sp. DA3-7 exhibited antimicrobial activity against Enterococcus
faecalis and Salmonella typhimurium, with minimum inhibitory concentrations of 78 and 156 μg/mL, respectively,
as well as strong cytotoxicity (24 h IC50 85 μg/mL) against MCF-7 human breast adenocarcinoma cells. The
active compound was separated, purified, and identified as Pyridine-2,5-diacetamide (C9H11N3O2 + H+,
194.21), which possessed a lowest minimum inhibitory concentration (31.25 μg/mL) against both Escherichia
coli and Cryptococcus neoformans. The antimicrobial activities of this novel compound are reported here for the
first time.

1. Introduction
Actinobacteria provide an unlimited source of bioactive compounds
that can be used for a variety of applications, and the genus
Streptomyces, in particular, has yielded several important bioactive
compounds, including antibiotics, bio-herbicides, and enzymes, all of
which possess high commercial value. In fact, nearly two-thirds of the
antibiotics known today have been obtained from Streptomyces.
However, even though the number of antimicrobial compounds ob-
tained from this genus increased exponentially each year between 1960
and 1970, the identification of such compounds progressed at a slower
pace during the 1980s and 1990s (Watve et al., 2001).
This declining trend in the discovery of new antimicrobial com-
pounds and the enigmatic development of antibiotic resistance in bac-
teria has increased the need for scientists to investigate unexplored
habitats, in order to identify novel actinobacterial isolates and bioactive
compounds. Recently, researchers have focused on extremophilic
actinobacteria with the hope that these organisms would add an in-
novative dimension to antimicrobial natural products research (Zitouni
et al., 2004; Vijayakumar et al., 2012; Dhanasekaran et al., 2014), and
as a result, bioactive compounds have been identified in actinobacterial
isolates from the Algerian Saharan desert (Badji et al., 2006), Atacama
desert (Rateb et al., 2011), Egyptian desert (Koberl et al., 2011), Qin-
ghai-Tibet Plateau (Ding et al., 2013), and Thar desert (Thumar et al.,
2010).
In Saudi Arabia, more than 90% of the land area is occupied by
desert. However, the diversity of actinobacteria in this habitat and the
bioactive compounds that they ostensibly produce have been poorly
explored (Atta et al., 2010; Ara et al., 2012; Nithya et al., 2015).
Therefore, the aim of the present study was to screen the soil of Saudi
Arabian deserts for actinobacteria, with a special emphasis on a
bioactive compound produced by the desert actinobacterium Strepto-
myces sp. DA3-7.

⁎
Corresponding author at: Department of Microbiology, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620024, India.
E-mail addresses: dhansdd@gmail.com, ddhanasekaran@bdu.ac.in (D. Dhanasekaran).

https://doi.org/10.1016/j.micres.2017.11.012
Received 10 July 2017; Received in revised form 31 October 2017; Accepted 18 November 2017
Available online 21 November 2017
0944-5013/ © 2017 Elsevier GmbH. All rights reserved.
K. Nithya et al. Microbiological Research 207 (2018) 116–133

Fig. 1. Colony morphology and microscopic view of Streptomyces sp. DA3-7. (a) Isolate DA3-7 grown on ISP-2 medium; (b) Phase contrast microscopic view of isolate DA3-7.

Table 1 et al., 1996). The soil samples were serially diluted by aseptically
Morphology of isolate DA3-7 on different media. adding 10 g of soil in 90 mL of sterile distilled water (10−1), mixed by
shaking and further tenfold dilutions were made up to 10−6. The
Media Aerial mycelium Substrate mycelium Diffusible pigment Growth
0.5 mL aliquots of each soil, from the dilutions 10−4, 10−5 and 10−6
ISP 1 Greyish white Pale yellow − +++ were spread over the agar plates in triplicates and incubated at 28 °C for
ISP 2 Ash grey Brown − +++ 15 days. Three replicates were used for each dilution and the average
ISP 3 Ash grey Golden yellow − ++ colony count of actinobacteria formed on a plate was calculated. Data
ISP 4 Ash grey Greyish white − +++
ISP 5 Ash grey Greyish white − +++
were expressed as a colony forming units (CFU/g) of soil dry weight.
ISP 6 Pale yellow Pale yellow − + Colonies were recognized by their cultural and morphological features,
ISP 7 Ash grey Ash grey − +++ the actinobacterial isolates were purified. Stock cultures of these iso-
SCA Yellowish grey Pale yellow − +++ lates were maintained in cryotubes with 1.5 mL 20% (w/v) sterile
NA Pale yellow Pale yellow − +
glycerol solution at −20 °C (Wellington and Williams, 1978).
GYEA Greyish white Brown − +++

+++, good; ++, moderate; +, poor; −, absent. ISP, International Streptomyces Project; 2.2. Primary screening
GYEA, glucose yeast extract agar; NA, nutrient agar; SCA, starch casein agar.

Pathogenic bacteria and fungi were obtained from the American

2. Materials and methods
2.1. Isolation of actinobacteria
The chemicals and media used for the experimental analysis were of
analytical reagent grade and are purchased from Himedia (Mumbai,
India) and Sigma-Aldrich (Bengaluru, India). Soil samples were col-
lected from 10 different locations in the desert of the Riyadh Province
(24° 49′01.3″N; 46° 43′01.2″E), Saudi Arabia. Starch casein agar (SCA)
medium that was supplemented with amphotericin B (20 μg/mL) and
nalidixic acid (10 μg/mL) was used to isolate the actinobacteria (Ellaiah
type culture collection (ATCC; Virginia, USA) (Table 2). Bacterial and
fungal inocula were prepared using Mueller-Hinton (MH) broth and
Sabouraud dextrose (SD) broth, respectively, the antimicrobial activ-
ities of the actinobacterial isolates were assessed using the cross streak
method (Egorov, 1985).
2.3. Characterization of isolate DA3-7
The morphological characteristics of isolate DA3-7 was examined as
recommended by the International Streptomyces Project (ISP). The
growth, colour of the aerial and substrate mycelia, and the production

Table 2
Minimum inhibitory concentrations of the DA3-7 extract against various pathogenic microorganisms.

S. No. Test organism Strain number MIC

DA3-7 extract (mg/mL) Control (μg/mL)

Bacteria Streptomycin
1. Klebsiella pneumoniae ATCC 12882 0.312 25
2. Enterococcus faecalis ATCC 49532 0.078 12.5
3. Escherichia coli ATCC 10536 0.312 50
4. Proteus vulgaris ATCC 33420 0.312 25
5. Salmonella typhimurium ATCC 13311 0.156 50
6. Pseudomonas aeruginosa ATCC 27883 0.312 50
7. Staphylococcus aureus ATCC 6538P 0.312 25
Fungi Ketoconazole
8. Candida albicans ATCC 2091 0.625 25
9. Cryptococcus neoformans ATCC 90113 0.625 50
10. Saccharomyces cerevisiae ATCC 9763 0.625 25

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K. Nithya et al.
of diffusible pigments were observed and documented. In addition, the
effects of pH, temperature, and NaCl concentration were recorded, and
the biochemical properties of the isolate were studied, as well. To in-
vestigate patterns of antibiotic sensitivity, utilization of various carbon
and nitrogen sources were employed using the method described by
Shirling and Gottlieb (1966).
Finally, in order to characterize the isolate’s phylogenetic place-
ment, genomic DNA was extracted using the MN DNA extraction kit
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Microbiological Research 207 (2018) 116–133
Fig. 2. Cytotoxicity of the ethyl acetate extract of
isolate DA3-7. (a,b) Untreated MCF-7 human breast
adenocarcinoma cells; (c,d) Extract-treated MCF-7
cells; (a,c) MCF-7 cells after acridine orange and
ethidium bromide staining; (b,d) MCF-7 cells after
Hoechst 33258 staining; (e) 3-(4,5-di-methylthiazol-
2-yl)2, 5-diphenyl2-H-tetrazolium bromide assay,
inhibitory concentration of the DA3-7 extract against
MCF-7 cells; (f) relative percentages of apoptotic and
necrotic cells; and (g) relative percentage of cells
with abnormal nuclei. Scale bars indicate 5 μm. *,
significant difference (p < 0.05) when compared
with the control. (For interpretation of the references
to colour in this figure legend, the reader is referred
to the web version of this article.)
(Macherey-Nagel, Düran, Germany), following the manufacturer’s
protocol. The 16S rRNA gene was PCR-amplified according to Hong
et al. (2009) using an Eppendorf thermal cycler, with gene-specific
primers (27f: 5′-AGTTTGATCCTGGCTCAG-3′; 1492r: 5′-ACGGCTACCT
TGTTACGACTT-3′). The amplified products were subjected to agarose
gel electrophoresis (1.2%) and purified using the MN DNA purification
kit. The purified PCR products were then sequenced using Roche GSFLX
454 technology (Macrogen, Republic of Korea), and the resulting
K. Nithya et al. Microbiological Research 207 (2018) 116–133

Table 3 incubated at either 37 °C (bacteria) or 28 °C (fungi), and the zone of

Gas chromatography–mass spectroscopy (GC–MS) spectral profile of the DA3-7 extract. inhibition for each of the microbes was recorded. Streptomycin (10 μg/
disc) and ketoconazole (25 μg/disc) were used as positive controls for
S. No. Retention Name of the Base (m/z) Area (%) Similarity (%)
time (min) compound the bacteria and fungi, respectively, and DMSO was used as a negative
control.
1. 11.28 Taxicatigenin 154.20 6.25 71 In addition, the MIC of the DA3-7 extract was determined for each
2. 12.00 Phthalic acid 149.15 3.96 65
of the pathogenic bacteria and fungi (Andrews, 2001). To accomplish
3. 12.50 Hexahydro-2H- 154.20 7.39 71
pyrido(1,2-a) this, the extract was dissolved in DMSO (100 mg/mL), and a two-fold
pyrazin-3(4H) dilution series (2500, 1250, 625, 312, 156, 78, and 39 μg/mL)was
4. 12.72 Pyrrolo[1,2-a] 154.20 19.99 91 prepared and loaded onto microtiter plates, in which each well con-
pyrazine-1,4- tained 100 μL of Mueller Hinton broth and Sabouraud dextrose broth
dione
for bacteria and fungi, respectively. Then, 5 μL of individual pathogen
5. 12.82 2(3H)-Furanone 154.20 7.53 76
6. 13.07 Hexadecanoic 129.25 2.29 87 cultures was inoculated to each well at a final inoculum density of
acid 1 × 105 cfu/mL. The inoculated plates were incubated at 37 °C (bac-
7. 13.16 Dibutyl phthalate 149.15 4.53 63 teria) and 28 °C (fungi) for 24 h. Streptomycin and ketoconazole were
8. 15.43 2,5- 170.15 17.52 96
used as positive controls for the bacteria and fungi, respectively, and
Piperazinedione
9. 16.21 Benzaldehyde 152.20 2.72 61 DMSO was used as a negative control.
10. 17.31 Ergotaman-3′,6′ 125.25 2.06 79 In order to determine the cytotoxicity of the DA3-7 extract, MCF-7
human breast adenocarcinoma cells, which were obtained from the
National Centre for Cell Science (Pune, India). The DA3-7 extract was
dissolved in DMSO, in order to prepare a range of extract concentra-
tions (10–100 μg/mL), and the solutions were loaded onto 96-well
plates, in which each well contained 5 × 103 MCF-7 cells and 200 μL
fresh medium. DMSO was used as a negative control. After 24 and 48 h
of incubation, a miniaturized viability assay was conducted (Mosmann,
1983) and the absorbance was recorded at 570 nm (measurement) and
630 nm (reference), using a microplate reader (iMark, Bio-Rad). The
Fig. 3. Structure of the bioactive compound (Pyridine-2,5-diacetamide) isolated from
assays for each concentration were performed in triplicate, and the
Streptomyces sp. DA3-7 extract. average percent inhibition was calculated as follows:
ODuntreated − ODtreated
Percent inhibition(%) = × 100.
Table 4 ODuntreated
Characteristics of Pyridine-2,5-diacetamide.
Both the treated and control MCF-7 cells were stained with 25 μL
S.No. Characters Results acridine orange (3.8 μM, in PBS) and ethidium bromide (2.5 μM, in
PBS), and the cell viability, nuclear morphology, membrane integrity,
1. Colour Light yellow
and percentages of apoptotic and necrotic cells were observed using a
2. Odour Pungent, irritating odour
3. Lustre Shiny fluorescent microscope (Carl Zeiss) (Spector et al., 1998). The cells
4. Stability Store in dark place were also stained with Hoechst 33258 (1 mg/mL, aqueous), and pa-
5. Solubility Ethanol, methanol, DMSO, water thological changes in nuclear morphology were observed using a
6. Melting point 110 °C fluorescent microscope (Latt et al., 1975).
7. pH stability 4.5–8.0
Finally, in order to determine the composition of the DA3-7 extract,
8. Temperature stability Up to 45 °C
9. IR (cm−1) 3417.16, 1706.59, 1388.62, 1254.75, 1011.97, gas chromatography (GC) and mass spectrometry (MS) were performed
885.42 using a 5890 GC that was equipped with a 5973 MS (Agilent
1
10. H NMR(ppm) 8.596, 7.455, 3.201, 1.940 Technologies) and a Triple-Axis HED-EM detector (Agilent
13
11. C NMR (ppm) 173.9, 149.18, 138.38, 125.24, 49.36, 21.64
Technologies). Helium was used as a carrier gas at 1mKL/min, and the
12. Mass spectrum (m/z) 194.19
13. Molecular formula C9H11N3O2 HP-5MS column dimensions were 30.0 m × 0.25 m × 0.10 μm. The
compounds in the DA3-7 extract were identified by comparison to the
NIST 11 library.
sequences were submitted to GenBank. The similarity index was per-
formed using BLASTn, and a phylogenetic tree was constructed using 2.5. Purification of bioactive compound
MEGA v6.0 (Tamura et al., 2013).
Batch submerged fermentation process was carried out using opti-
2.4. Crude extract characterization mized MNG medium (3 g/L peptone, 5 g/L beef extract, 1 g/L yeast
extract, 5 g/L glucose, and 2 g/L NaCl, pH 7, 120 rpm, 4% inoculum,
Isolate DA3-7 was grown in five different fermentation media at 30 °C for 8 d) for mass-scale production of bioactive compounds from
28 °C in a rotary shaker at 120 rpm for 8d. The fermented broth was the isolate DA3-7. After fermentation, the supernatant from culture
tested against the pathogenic microorganisms by well-diffusion filtrate was extracted with an equal volume of ethyl acetate and con-
method. A 4% DA3-7 inoculum was introduced to 700 mL of selected centrated using a rotary evaporator. The crude DA3-7 extract (2 g) was
MNG medium and incubated at 28 °C in a rotary shaker (120 rpm) for purified using column chromatography with a silica gel column
8d. The broth was centrifuged, and the supernatant was extracted with (25 × 5 cm), silica gel (60 mesh size, Merck, India), and a gradient
equal volume of ethyl acetate and concentrated using a rotary eva- solvent system of chloroform and methanol (8:2, 7:3, 6:4, 5:5, 4:6, 3:7,
porator, and then used for pharmacological analysis. and 2:8). The collected fractions was assessed their antimicrobial po-
The antimicrobial activity of the DA3-7 extract was assessed using tential against P. vulgaris, using the disc diffusion method (Bauer et al.,
the disc diffusion method (Bauer et al., 1966). The sterile disc, filled 1966). Finally, the active fractions were pooled together and analysed
with 0.5 mg/disc concentration of the DA3-7 extract, was prepared using an HPLC (Shimadzu) equipped with a binary pump, a UV de-
using DMSO. The discs were placed over the inoculated plates and tector, an auto sampler, and a column thermostat. The chromatography

119
K. Nithya et al.
separations conducted using a DL-C18 column (5.0 μm, 250 × 4.6 mm)
at 30 °C, and elution was performed using a flow rate of 0.5 mL/min at
260 and 300 nm with methanol and water as the mobile phase.
2.6. Characterization of bioactive compound
The bioactive compound of isolate DA3-7 was subjected to Fourier
transform and infra-red (FT-IR) spectrum analysis, using a FT-IR Alpha
II (Bruker Corp., MA, USA) instrument that was equipped with AT-XT
Golden gate accessories. The FT-IR spectrum of active compound was
also scanned in the 400–4000 cm−1 range and then the spectrum was
plotted as intensity versus wave number (Augustine et al., 2005). The 1H
NMR, 13C NMR, and 13C DEPT-NMR spectra were measured in DMSO d6
using an NMR GSX-400 (JEOL Ltd., Tokyo, Japan) spectrophotometer
at 400 MHZ for 1H and 300 MHZ for 13C. All the NMR spectra were
attained at 25 °C and also the spectral acquired with 4000–8000 scans.
Further, the data were administered with exponential decay equivalent
to 1 Hz line broadening. The acquisition parameters for all the NMR
spectral analysis were presented in Figs. A9 and A10. Acetone and
methanol d4 was used as the internal standard for 1H NMR and 13C
NMR, respectively. The mass spectra were recorded using a mass
spectrophotometer Finnigan MAT 8230 (Thermo Scientific, NY, USA).
The molecular structure of the bioactive compound was then elucidated
from the FT-IR, 1H NMR, 13C NMR, 13C DEPT-NMR, and mass spectra.
The molecular formula and molecular weight of the compound were
also determined.
The solubility, temperature stability and pH stability of the bioac-
tive compound was tested. Finally, the MICs of the bioactive compound
against a variety pathogenic bacteria and fungi were determined, fol-
lowing the guidelines of British Society of Antimicrobial Chemotherapy
(BSAC) (Andrews, 2001). To accomplish this, the compound was dis-
solved in DMSO (10 mg/mL), and a two-fold dilution series (250, 125,
62.5, 31.25, 15.62, 7.81, 3.90, and 1.95 μg/mL) was prepared. The
detailed MICs protocol was described in the Section 2.4.
2.7. Statistical analysis
The data were statically analysed using the SPSS statistical analysis
software (v 11.5), and the results were expressed as means ± standard
deviation (SD). One-way analysis of variance (ANOVA) was used to
analyse cytotoxicity.
3. Results
3.1. Actinobacteria isolation and screening
A total of 134 morphologically distinct actinobacterial isolates were
obtained from 10 different desert soil samples, and preliminary
screening (cross streak method) revealed that only 16 of the isolates
possessed antimicrobial activity. Among these, one isolate (DA3-7)
possessed broad-spectrum antimicrobial activity against both gram-
positive and gram-negative bacteria, as well as against fungi.
3.2. Characterization and identification of isolate DA3-7
Isolate DA3-7 formed a whitish, ash-coloured aerial spore mass on
ISP-2 medium (Fig. 1a) and produced hook-like sporophores that ex-
tended to form spirals, with more than 10 spores per chain (Fig. 1b).
The isolate grew well in all the tested media, on which it produced grey
to whitish-grey aerial mycelia, but ISP-2 medium the most suitable (Fig.
A1). Diffusible pigments were not observed in any of the tested media.
The substrate mycelia of isolate DA3-7 were brown and brick red in
colour in the ISP-2 and GYEA media, respectively, whereas the sub-
strate mycelia were pale yellow or ash-coloured in the other media
(Table 1). The morphology of the DA3-7 isolate indicated that it was
closely related to the genus Streptomyces.
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Microbiological Research 207 (2018) 116–133
The isolate was able to tolerate temperatures of up to 40 °C, pH
conditions of up to 11, and NaCl concentrations of up to 3% and was
unable to grow at 45 °C, pH conditions from 3 to 5, and NaCl con-
centrations from 4 to 5%. Isolate DA3-7 produced positive results in
methyl red, citrate, urease, catalase, and amylase, but negative results
were obtained for gelatinase, protease, and lipase. Isolate DA3-7 was
sensitive to six of 15 tested antibiotics and was resistant to the re-
maining eight (Table A1). Of nine different carbon sources, isolate DA3-
7 fully utilized seven but only partially utilized arabinose and maltose.
Of 10 different nitrogen sources, only alanine, glycine, L-leucine, and L-
lysine monohydrochloride fully supported the growth of the isolate,
whereas L-cystine, L-histidinemonohydrochloride, DL-methionine, and
L-proline only provided moderate support, and DL-aspartic acid and L-
glutamic acid provided no support (Table A2).
In addition, the isolate’s 16S rDNA gene sequence, which was am-
plified from its genomic DNA, was ∼1600 bp long. BLASTn analysis
indicated that isolate DA3-7 was closely related to Streptomyces sp.,
with 100% similarity. The 16S rRNA gene sequence of isolate DA3-7
was submitted to the GenBank (NCBI) under the accession number
KT365284. The phylogenetic relationship was studied between the 16S
rRNA gene sequence of the isolate DA3-7 and other closely related gene
sequences available in the GenBank database. The unrooted phyloge-
netic tree revealed that the isolate DA3-7 was closely related to
Streptomyces sp. GA-5 (KJ139432), as they formed a distinct clade (Fig.
A2).
3.3. Bioactive compound production, extraction, and antibiogram
The selection of an appropriate media is an important prerequisite
for culturing actinobacteria to produce antimicrobial compounds. In
this study, five different fermentation media were screened for anti-
microbial compound production. Among them, modified nutrient glu-
cose (MNG) medium was the most suitable for the production of anti-
microbial compounds by Streptomyces sp. DA3-7 (Fig. A3). Therefore,
the fermentation process of the isolate DA3-7 was in MNG medium, and
the broth was extracted using ethyl acetate. The resulting crude extract
also possessed promising antimicrobial activity against the bacterial
and fungal pathogens, with zones of inhibition ranging from 10 to
13 mm and from 8 to 18 mm, respectively (Fig. A4).
3.4. Minimal inhibitory concentration (MIC), cytotoxicity and GC–MS
analysis of the DA3-7 extract
The MIC study indicated that the extract possessed activity against
the bacteria Enterococcus faecalis (0.078 mg/mL), Salmonella typhi-
murium (0.156 mg/mL), Klebsiella pneumoniae (0.312 mg/mL),
Escherichia coli (0.312 mg/mL), Proteus vulgaris (0.312 mg/mL),
Pseudomonas aeruginosa (0.312 mg/mL), and Staphylococcus aureus
(0.312 mg/mL), and the fungi (0.625 mg/mL) Candida albicans,
Cryptococcus neoformans, and Saccharomyces cerevisiae. The MICs for E.
faecalis and S. typhimurium were highly significant (Table 2).
An in vitro cytotoxicity assay was performed for different con-
centrations (10–100 μg/mL) of the DA3-7 extract against MCF-7 breast
adenocarcinoma cells, and the results are presented in Fig. 2a–g. After
24 h, the IC50 was 85 μg/mL (Fig. 2e), and the percentages of apoptosis
and necrosis were 45 and 7%, respectively (Fig. 2f). In addition,
Hoechst 33528 staining revealed that 42% of the treated cells had ab-
normal nuclei (Fig. 2g).
GC–MS of the DA3-7 extract identified 10 compounds (Table 3 and
Fig. A5). Among them, pyrrolo[1,2-a]pyrazine-1,4-dione and 2,5-pi-
perazinedione constituted 19.99 and 17.52% of the extract, respec-
tively. The other compounds were identified as 2(3H)-furanone
(7.53%), hexahydro-2H-pyrido(1,2-a)pyrazin-3(4H) (7.39%), taxi-
catigenin (6.25%), dibutyl phthalate (4.53%), phthalic acid (3.96%),
benzaldehyde (2.72%), hexadecanoic acid (2.29%), and ergo-
taman-3′,6′ (2.06%).
K. Nithya et al.
3.5. Purification of bioactive compounds
Optimized MNG medium was used for the extraction of bioactive
compounds from Streptomyces sp. DA3-7. Bioactive compounds were
then extracted from the fermented broth and purified. The crude DA3-7
extract was subjected to column chromatography using the chlor-
oform:methanol solvent system, with eluent conditions of 8:2, 7:3, 6:4,
5:5, 4:6, 3:7, and 2:8, which yielded 18 fractions. In total, six of the
fractions (2, 3, 4, 5, 6 and 7) were active against P. vulgaris (Fig. A6a–b).
All the active fractions were pooled and further purified using high-
performance liquid chromatography (HPLC) at 296 and 300 nm (Fig.
A7a–b). Based on the maximum area percentages, four of the com-
pounds were selected for further analysis and categorized as F1 (3.872
RT at 300 nm), F2 (3.889 RT at 296 nm), F3 (6.397 RT at 296 nm), and
F4 (6.177 RT, 300 nm). Compound F1 exhibited good antimicrobial
activity against both pathogenic bacteria (E. coli (25 mm), P. aeruginosa
(20 mm), S. aureus (19 mm) and S. typhimurium (19 mm)) and fungi (C.
neoformans (24 mm), C. albicans (17 mm), and S. cerevisiae (14 mm)),
whereas F2 exhibited poor antimicrobial activity, and compounds F3
and F4 failed to exhibit any activity against the pathogenic micro-
organisms (Table A3). Hence, compound F1 was selected for structure
elucidation.
3.6. Characterization of bioactive compound F1
Compound F1 was completely soluble in dimethylsulphoxide
(DMSO), and the IR spectrum of compound F1 was mainly characterized
by strong peaks at 3351, 1677, 1640, 1399, 1277, 1016, and 676 cm−1
which are attributed mainly to the stretching frequencies of eNH, eC]
N, eC]O, and −OeCeR (R, alkyl group) groups. The strong peaks in
the spectrum at 3351 cm−1 indicate the presence of −NH stretching in
the compound. The broad peak at 1677, 1399 cm−1 is due to eC]O
stretching while peak at 1640 cm−1 indicates the presence of amine
group. The strong peak at 1277, 1016 cm−1 is assignable to stretching
frequencies of eOeCe moiety in the molecule. The peak appeared in
the region < 1000 cm−1 indicates the presence of eCeO, eCeN bond
in the molecule (Fig. A8).
Meanwhile, the 1H NMR spectrum of the compound, which was
recorded in a DMSO-d6 solution, with tetramethylsilane as an internal
standard, indicated that different types of proton signal, appeared in the
spectrum of the compound indicating different chemical environment.
In addition, the molecule actually exhibited keto-enol tautomerism in
solution, and the characteristic signal (doublet) at ∼8.5 ppm indicated
the proton of an aromatic amide group (-CONH). Other signals in the
aromatic region indicate the presence of protons attached to pyridine
ring. The proton signals at 4.0 ppm come from solvent. Characteristic
proton signals ∼2.0 ppm account for the presence of methyl group
attached to carbonyl system (Fig. A9). The assignment of protons in the
molecular structure was confirmed using 13C NMR (Fig. A10a–b). The
assignment of proton in the molecular structure is further consolidated
by 13C NMR and ESI–MS spectrum proves in favour of this molecular
composition. This 13C NMR spectral pattern agrees very well with the
proposed molecular structure and proton signal shift in 1H NMR. ESI-Ms
consolidates the structural existence of the proposed molecule. In the
ESI mass spectrum (Fig. A11), the characteristic peak is found at m/z
78.2046 (C5H3N, 78.04) as base peak while other characteristic peaks
at m/z 93.2025 (C5H4N2 + H+, 93.05), 136.2078 (C7H8N2O + H+,
136.15), 152.2346 (C7H9N3O + H+, 152.07), and 194.1910
(C9H11N3O2 + H+, 194.21) strongly supports for the existence of the
proposed molecule. On the basis of the spectral data, the structure of
compound F1 was elucidated as Pyridine-2,5-diacetamide (Fig. 3), with
a molecular formula of C9H11N3O2 + H+.
The bioactive compound Pyridine-2,5-diacetamide is light yellow in
colour, pungent, with an irritating odour, and shiny in nature. The
compound was soluble in ethanol, methanol, DMSO, and water, and the
melting point of the compound was 110 °C. In addition, the compound
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Microbiological Research 207 (2018) 116–133
was stable between pH 4.5 and 8.0 and up to 45 °C. The details of the
spectral profiles, molecular weight, and molecular formula of the
compound are presented in Table 4.
3.7. MIC of Pyridine-2,5-diacetamide
MIC of Pyridine-2,5-diacetamide was determined, the compound
showed activity against all the examined pathogenic bacteria, with MIC
values ranging from 31.25 to 125 μg/mL. The lowest MIC value was
exhibited against E. coli (31.25 μg/mL), followed by those against S.
typhimurium (62.5 μg/mL), S. aureus (62.5 μg/mL), P. vulgaris (62.5 μg/
mL), P. aeruginosa (62.5 μg/mL), E. faecalis (62.5 μg/mL), and K.
pneumoniae (125 μg/mL). However, the compound’s MIC against C.
neoformans (31.25 μg/mL) was lower than that of ketoconazole (50 μg/
mL), and the compound also inhibited the growth of C. albicans and S.
cerevisiae at 62.5 μg/mL (Table A4 and Fig. A12).
4. Discussion
Actinobacteria from unexplored ecosystems, especially desert en-
vironments, represent a potentially useful source of bioactive metabo-
lites and have the potential for biotechnological applications. In the
present study, a total of 134 actinobacterial isolates were obtained from
desert soil samples, and of these, 16 isolates exhibited antimicrobial
potential. These actinobacteria are, therefore, capable of producing
highly bioactive metabolites and provide scope for the discovery of
novel drugs. Most secondary metabolites and antibiotics from actino-
bacteria are extracellular in nature (Saravana Kumar et al., 2014). In
the present study, Streptomyces sp. DA3-7 produced extracellular sec-
ondary metabolites, which were extracted using ethyl acetate and
which exhibited respectable antimicrobial activity against various pa-
thogenic bacteria and fungi. Many previous studies have reported that
ethyl acetate is a suitable solvent for extracting antimicrobial com-
pounds from actinobacteria and, particularly, from the genus Strepto-
myces (Duraipandiyan et al., 2010; Vijayakumar et al., 2012; Saravana
Kumar et al., 2014).
Actinobacteria can produce a variety of bioactive natural products,
and this potential is so great that there is a continuous search for novel
therapeutics and preventive agents for controlling cancer and diabetes
(Ser et al., 2016). Apoptotic cell death is characterized by different
cellular changes, including cell shrinkage, nuclear condensation, DNA
fragmentation, membrane blebbing, and the formation of apoptotic
bodies. These cytological changes were observed in the MCF-7 cells
treated with the DA3-7 extract. The acridine orange- and ethidium
bromide-stained cells were classified into four types: i) viable cells with
highly organized and green-fluorescent nuclei (Fig. 2a, b), ii) early
apoptotic cells with condensed and orange-green fluorescent nuclei
(Fig. 2c), iii) late apoptotic cells with highly condensed or fragmented
chromatin and orange to red fluorescence (Fig. 2c), and iv) swollen
necrotic cells with no indication of chromatin fragmentation and or-
ange to red fluorescence. Hoechst 33528 staining method also indicated
that the nuclei of DA3-7 extract-treated MCF-7 cells exhibited mor-
phological changes, and after 24 h, the treated cells exhibited chro-
matin marginalization and nuclear condensation and fragmentation
(Fig. 2d). Thus, the in vitro cytotoxicity screening demonstrated the
presence of biologically active metabolites in the DA3-7 extract.
The crude DA3-7 extract should be studied further, in order to im-
prove its purification and characterization, to confirm the identity of its
constituent active secondary metabolites, and to bring up economically
valuable compounds towards natural products discovery. In the present
study, fermentation of the optimized MNG medium was performed by
Streptomyces sp. DA3-7. The cell-free culture filtrate was extracted and
concentrated, and the crude compounds were purified and character-
ized. The main active compound was identified as Pyridine-2,5-diace-
tamide, which has a molecular formula of C9H11N3O2 + H+ bears no
resemblance to other active compounds reported in databases or
K. Nithya et al. Microbiological Research 207 (2018) 116–133

literature (e.g., http://chembiofinder.cambridgesoft.com and www.
chemspider.com), to other antibiotics described in the Dictionary of
antibiotics and related substances (Bycroft, 1988), in the Dictionary of
natural products (Buckingham, 1997), or in a review of bioactive mi-
crobial metabolites (Berdy, 2005). Thus, Pyridine-2,5-diacetamide is
apparently an pyridine alkaloid hitherto unreported from plants, fungi,
algae, bacteria, or actinobacteria.
Several bioactive compounds have been reported from Streptomyces,
including neomycin B and C (Sambamurthy and Ellaiah, 1974), mon-
ensins A and B (Beran and Zima, 1993), 2-methyl-heptyl isonicotinate
(Bordoloi et al., 2001), arylomycins A and B (Schimana et al., 2002),
himalomycin A and B (Maskey et al., 2003), 4-phenyl-1-napthylphe-
nylacetamide (Dhanasekaran et al., 2008), N-phenylpropanamide
(Priyadharsini et al., 2013), 1,5,7-trihydroxy-3-hydroxy methyl an-
thraquinone (Duraipandiyan et al., 2014). It is interesting to note that
pyridine alkaloids has recently been identified in Streptomyces species
(Groenhagen et al., 2014; Zohu and Fenical, 2016; Betancur et al.,
2017), and the present study reports that the desert actinobacterium
Streptomyces sp. DA3-7 produces a novel pyridine alkaloid (Pyridine-
2,5-diacetamide).
The MICs of Pyridine-2,5-diacetamide against pathogenic bacteria
and fungi were lowest against the bacterium E. coli (31.25 μg/mL) and
the fungi C. neoformans (31.25 μg/mL), when compared to the standard
antibiotics streptomycin and ketoconazole. Antimicrobial compounds
produced by Actinomadura sp. AC104 isolated from Algerian Saharan
soil and which exhibited more antifungal activity (Badji et al., 2006).
Bioactive ansamycins produced by a Streptomyces sp. from a hyper-arid
desert (Salar de Atacama) exhibit promising antibacterial activity
against methicillin-resistant S. aureus (Rateb et al., 2011). Boubetra
et al. have also reported two novel compounds from Saccharothrix
SA198, isolated from Saharan desert soil, and these antibiotic com-
pounds exhibit strong activity against Mucor ramannianus and Asper-
gillus carbonarius and moderate activities against both gram-positive
and gram-negative bacteria (Boubetra et al., 2013).
5. Conclusion
Taken together, the results of the present study demonstrate that the
Saudi Arabian desert soil actinobacterium Streptomyces sp. DA3-7 pro-
duces a novel bioactive compound (Pyridine-2,5-diacetamide) that ex-
hibits broad-spectrum antimicrobial activity against pathogenic bac-
teria and fungi. In the present study concludes that the actinobacteria
are ubiquitous, they vary in their morphological, physiological, bio-
chemical, cultural, and molecular characteristics depending on the
physico-chemical properties of the habitats. The desert environment
possessed more potential, rare and novel actinobacterial communities,
and the extensive studies need for isolations, innovative and effective
taxonomic approaches which could be lead to the discovery of novel
genera and novel species of actinobacteria. The increase of antibiotic-
resistant bacteria and declining trend in the discovery of new anti-
microbial compounds have upturned pressure on the scientist, it is the
time to investigate unexplored habitats especially in desert environs for
the discovery of novel bioactive compounds, which could be useful to
the society.
Competing interests
The authors declare that no competing financial interests.
Ethical approval
Not required.
Acknowledgements
K.N. is grateful to the University Grant Commission (UGC),
Government of India for an RGNF-SRF award (UGC approval no.: F1-
17.1/2011-12/RGNF-SC-TAM-1376), and D.D. is grateful to the
University Grant Commission (UGC), Government of India for financial
support, in the form of a Raman Fellowship for Post-Doctoral Research
in the USA (F.no: 5-29/2016(IC), dated 10 February 2016). The authors
would also like to extend their sincere appreciation to the Deanship of
Scientific Research at King Saud University for funding this work
through Research Group No. RG-1438-074.

Appendix A

Table A1
Characteristics and antibiotic sensitivity of isolate DA3-7.

Characteristics Results Antibiotic disc Results

Gram’s stain Positive Cefuroxime R

Shape and growth Filamentous aerial growth Cefpodoxime R
Growth temperature 25–40 °C Moxifloxacin S
Growth pH 5–10 Piperacillin R
NaCl tolerance 1–3% Streptomycin S
Indole − Levofloxacin S
Methyl red + Lincomycin R
Voges-Proskauer − Ampicillin R
Citrate utilization + Erythromycin R
Triple sugar iron agar − Norfloxacin R
Urease + Gatifloxacillin S
Catalase + Netillin S
Oxidase − Vancomycin S
Starch hydrolysis + Ofloxacin R
Gelatin hydrolysis − Penicillin R
Lipid hydrolysis −
Casein hydrolysis −

+, positive; −, negative; R, resistant; S, susceptible.

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Table A2
Utilization of carbon and nitrogen sources by isolate DA3-7.

Carbon and nitrogen sources Growth

Carbon sources
Control +
Arabinose ++
Dextrose ++++
Maltose +
Mannose ++++
Galactose ++++
Lactose ++
Starch ++++
Sucrose ++++
Xylose ++++
Nitrogen sources
Control +
Alanine ++++
DL-Aspartic Acid −
L-Cystine +++
L-Glutamic acid −
Glycine ++++
L-Histidinemonohydrochloride ++
L-Leucine ++++
L-Lysine monohydrochloride ++++
DL-Methionine +++
L-Proline +++

+++, good; ++, moderate; +, poor; −, absent.

Table A3
Antimicrobial activity of four high-performance liquid chromatography (HPLC) fractions against various pathogenic microorganisms.

S. No. Test organism Strain number Zone of inhibition (mm)

F1 F2 F3 F4

Bacteria
1 Klebsiella pneumoniae ATCC 12882 13 4 – –
2 Enterococcus faecalis ATCC 49532 18 3 – –
3 Escherichia coli ATCC 10536 25 5 – –
4 Proteus vulgaris ATCC 33420 17 2 – –
5 Salmonella typhimurium ATCC 13311 19 2 – –
6 Pseudomonas aeruginosa ATCC 27883 20 3 – –
7 Staphylococcus aureus ATCC 6538P 19 4 – –
Fungi
8 Candida albicans ATCC 2091 17 4 – –
9 Cryptococcus neoformans ATCC 90113 24 6 – –
10 Saccharomyces cerevisiae ATCC 9763 14 2 – –

F1–F4, HPLC fractions.

Table A4
Minimum inhibitory concentrations of Pyridine-2,5-diacetamide against various pathogenic microorganisms.

S.No. Test organisms Strain number MIC

Compound (μg/mL) Control (μg/mL)

Bacteria Streptomycin
1. Klebsiellapneumonia ATCC 12882 125 25
2. Enterococcus faecalis ATCC 49532 62.5 12.5
3. Escherichia coli ATCC 10536 31.25 50
4. Proteus vulgaris ATCC 33420 62.5 25
5. Salmonella typhimurium ATCC 13311 62.5 50
6. Pseudomonas aeruginosa ATCC 27883 62.5 50
7. Staphylococcus aureus ATCC 6538P 62.5 25
Fungi Ketoconazole
8. Candida albicans ATCC 2091 62.5 25
9. Cryptococcus neoformans ATCC 90113 31.25 50
10. Saccharomyces cerevisiae ATCC 9763 62.5 25

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Fig. A1. Colony morphology the isolate DA3-7

grown on different media. ISP, International
Streptomyces Project; GYEA, glucose yeast extract
agar; NA, nutrient agar.

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Fig. A2. Unrooted phylogenetic tree of the 16S rRNA gene sequence of Streptomyces sp. DA3-7 and other closely related gene sequences by Neighbor-joining method. Numbers in −the
parentheses indicate GenBank accession numbers.

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Fig. A3. Antimicrobial activity of DA3-7 culture broth derived from different media. (a) Cryptococcus neoformans and (b) Staphylococcus aureus. 1, ISP-2 medium; 2, M6 medium; 3, MNG
medium; 4, SD medium, 5, YPG medium.

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Fig. A4. Antimicrobial activity of the ethyl acetate extract of isolate DA3-7. (a) Klebsiella pneumoniae;(b) Enterococcus faecalis; (c) Candida albicans;(d) Escherichia coli; (e) Proteus vulgaris;
(f) Salmonella typhimurium;(g) Cryptococcus neoformans;(h) Pseudomonas aeruginosa;and (i) Staphylococcus aureus.CA, ethyl acetate extract of isolate DA3-7; NC, DMSO as a negative
control; S, streptomycin as a positive control for the bacteria; NS, nystatin as a positive control for the fungi.

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Fig. A5. Gas chromatography–mass spectroscopy (GC–MS) profile of the ethyl acetate extract of Streptomyces sp. DA3-7.

Fig. A6. a-b. Antibacterial activity of fractions de-

rived from DA3-7 extract against Proteus vulgaris;
1–18, fractions collected from DA3-7 extract by
column chromatography; SC, solvent control.

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Fig. A7. High-performance liquid chromatography of fractions derived from Streptomyces sp. DA3-7. (a, b) Chromatograms at 296 and 300 nm, respectively.

Fig. A8. Infra-red (IR) spectrum of compound F1 from Streptomyces sp. DA3-7.

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Fig. A9. 1H NMR spectrum of compound F1 from Streptomyces sp. DA3-7.

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13
Fig. A10. a. C NMR spectrum of compound F1 from Streptomyces sp. DA3-7. b. DEPT13C NMR spectrum of compound F1 from Streptomyces sp. DA3-7.

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Fig. A11. Mass spectrum of compound F1 from Streptomyces sp. DA3-7.
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