ORIGINAL PAPER

Streptomyces amritsarensis sp. nov., exhibitingbroad-spectrum antimicrobial activity

Deepika Sharma • Shanmugam Mayilraj •

Rajesh Kumari Manhas

Received: 31 October 2013 / Accepted: 10 March 2014 / Published online: 25 March 2014

� Springer International Publishing Switzerland 2014

Abstract A new actinobacterium strain, designated

2AT, was isolated from a soil sample collected from

Guru Nanak Dev University, Punjab (India) and

characterized using a polyphasic taxonomic approach.

It showed antimicrobial activity against various Gram-

positive and Gram-negative bacteria including drug

resistant bacteria and fungi. The strain had chemotax-

ononomic and morphological properties typical of the

genus Streptomyces. The 16S rRNA gene sequence of

the strain showed 99.9, 99.5 and 99.5 % similarity

with Streptomyces flavotricini DSM 40152T, Strepto-

myces toxytricini DSM 40178T and Streptomyces

globosus DSM 40815T, respectively. This strain

formed a coherent cluster with them and shared

DNA–DNA homology of 37.6 ± 0.6, 34.4 ± 0.5 and

33.1 ± 0.4 % with type strains, S. flavotricini DSM

40152T, S. globosus DSM 40815T and S. toxytricini

DSM 40178T, respectively. Further, the strain was

readily distinguished from the phylogenetic close

relatives in a variety of morphological, physiological

and biochemical properties. Based on the genotypic

and phenotypic characteristics, it is proposed that

strain 2AT represents a novel species in the genus

Streptomyces, for which the name Streptomyces

amritsarensis sp. nov. is proposed, with the type strain

2AT (=MTCC 11845T=JCM 19660T).

Keywords Actinobacterium � Streptomyces

amritsarensis sp. nov � Polyphasic taxonomy

Introduction

The genus Streptomyces, encompassing nearly 600

species with validly published names (Euzeby 2012),

is the largest genus in domain Bacteria. The genus,

first described by Waksman and Henrici (1943),

includes aerobic, Gram-positive, high G?C content

(69–78 mol%) bacteria producing extensively branch-

ing networks of substrate mycelia that give rise to the

vertical branching aerial hyphae bearing chains of

spores. Members of this genus are well known for their

production of antibiotics. However, to combat the

problem of microbial resistance and development of

new antimicrobials to substitute the ineffective ones, it

is imperative to search new, efficient and safe

antibiotics. To discover new therapeutic compounds

we have to screen phylogenetically novel streptomy-

cetes which will avoid the costly rediscovery of

already known compounds from Streptomyces species

Electronic supplementary material The online version ofthis article (doi:10.1007/s10482-014-0151-2) contains supple-mentary material, which is available to authorized users.

D. Sharma � R. K. Manhas (&)

Department of Microbiology, Guru Nanak Dev

University, Amritsar 143005, India

e-mail: rkmanhas@rediffmail.com

S. Mayilraj

Microbial Type Culture Collection and Gene Bank

(MTCC), CSIR-Institute of Microbial Technology

(IMTECH), Chandigarh 160036, India

123

Antonie van Leeuwenhoek (2014) 105:943–949

DOI 10.1007/s10482-014-0151-2

(Antony-Babu and Goodfellow 2008). In a screening

program for novel antimicrobial compounds from

actinobacteria, isolate 2AT which demonstrated potent

antimicrobial activity against various organisms

including drug resistant bacteria [methicillin resistant

Staphylococcus aureus (MRSA), vancomycin-resis-

tant Enterococcus (VRE) and multi drug resistant

E. coli] was isolated from a soil sample collected from

Guru Nanak Dev University, Amritsar, Punjab (India).

The present study was undertaken to establish the

taxonomic status of this strain. The polyphasic taxo-

nomic study showed that isolate 2AT belonged to a

new Streptomyces species, for which the name Strep-

tomyces amritsarensis sp. nov. is proposed.

Materials and methods

Microorganisms, maintenance and culture

conditions

Strain 2AT was isolated from a soil sample collected

from Guru Nanak Dev University, Punjab, India

(31.63�N, 74.87�E) using standard dilution plating

method. Soil sample was serially diluted up to 10-6

and 0.1 ml aliquot from each dilution was spread onto

starch casein nitrate agar supplemented with cyclo-

heximide (50 lg/ml) and nystatin (25 lg/ml, HiMe-

dia, India). The plates were incubated at 28 �C for

28 days (Sharma et al. 2011).

The isolate and the type strains of S. flavotricini

DSM 40152T, S. globosus DSM 40815T and S.

toxytricini DSM 40178T were maintained on modified

Bennett’s agar (Jones 1949) slopes at 4 �C and as

mycelial fragments and spores in 20 % v/v glycerol at

-80 �C.

Morphological, physiological and biochemical

characterization

Cultural characteristics were determined as per meth-

ods prescribed in International Streptomyces Project

(Shirling and Gottlieb 1966). The pigmentation of

aerial and substrate mycelia, soluble pigment and

sporulation which are highly characteristic and useful

in the classification of the genus Streptomyces were

observed by cultivating the strain on starch casein

nitrate agar, Bennett’s agar and different ISP media at

28 �C for 1 week. Morphological properties of the

strain were observed by light microscope (Olympus)

and spore surface was determined by scanning elec-

tron microscopy following desiccation of the cells and

gold coating, using a Carl Zeiss model EVOLS 10.

Physiological tests such as growth at different

temperatures (12–42 �C), pH (5.0–10.0) and NaCl

(1–7 %, w/v) concentrations were performed by

growing the strain on starch casein nitrate agar at

28 �C. Assimilation of sugars as carbon sources (1 %)

was studied according to Shirling and Gottlieb (1966).

D-Glucose, sucrose, xylose, inositol, D-mannitol, D-

fructose, L-rhaminose, D-raffinose and cellulose (1 %)

(HiMedia, India) were added to basal medium after

filter sterilization. Catalase production, citrate utiliza-

tion (Simmons) and urea hydrolysis were determined

according to Cowan and Steel (1965). Hydrolysis of

casein, gelatin and starch, indole production, MR-VP

and oxidase tests were performed as recommended by

Smibert and Krieg (1994). Nitrate reduction was

investigated according to Lanyi (1987).

Chemotaxonomic characterization

Freeze-dried cells for chemotaxonomic analysis

(except for fatty acid study) were prepared by

harvesting the mycelium in the late exponential phase

following growth in trypticase soy broth (TSB;

HiMedia, India) at 28 �C for 3 days. Analysis of

isomer of diaminopimelic acid in the cell wall and

sugars in the whole-cell hydrolysate was done accord-

ing to method given by Lechevalier and Lechevalier

(1980). Menaquinones were extracted and purified as

per method given by Saha et al. (2005). The purified

menaquinones were separated by reversed phase

HPLC (SCL-10AVP, Shimadzu) using the solvent

system of acetonitrile and isopropanol in a ratio of

65:35 with a flow rate of 1 ml/min and monitored at a

wavelength of 269 nm. For the determination of

cellular fatty acids, strain was grown on TSB medium

at 30 �C for 2 days; cellular fatty acids were extracted,

methylated and analysed using Gas Chromatography

according to the instructions of the Sherlock Microbial

Identification System (MIDI, USA Version 4.0; Ac-

tino library) as described previously (Sasser 1990;

Pandey et al. 2002). Extraction of polar lipids was

done based on the modified protocol of Bligh and Dyer

(1959). Two-dimensional TLC was run for identifica-

tion of polar lipids according to procedures described

by Komagata and Suzuki (1987). Lipid spots were

944 Antonie van Leeuwenhoek (2014) 105:943–949

123

detected using the following spray reagents: molyb-

datophosphoric acid (5 % w/v) in absolute ethanol,

molybdenum blue spray reagent (1.3 %, Sigma),

ninhydrin (0.2 % w/v) in acetone and anisaldehyde

reagent (Sigma) for detection of total lipids, phospho-

lipids, aminolipids and glycolipids, respectively.

Phylogenetic analysis and genomic relatedness

For 16S rRNA gene sequencing the genomic DNA

extraction and amplification was performed according

to the method described by Mayilraj et al. (2006).

Identification of phylogenetic neighbours and the

calculation of pairwise 16S rRNA gene sequence

similarities were achieved using the EzTaxon server

(http://eztaxon-e.ezbiocloud.net/; Kim et al. 2012).

Phylogenetic trees were constructed according to the

neighbour-joining and maximum-parsimony using

bootstrap values based on 1,000 replications with the

MEGA6 software (Tamura et al. 2013). The G?C

content of genomic DNA was determined spectro-

photometrically (Lambda 35, Perkin Elmer, Waltham,

MA, USA) using thermal denaturation method

(Mandel and Marmur 1968). DNA–DNA hybridiza-

tion was performed with freshly isolated genomic

DNA by the membrane filter method (Tourova and

Antonov 1987) and experiment was repeated twice.

Determination of antimicrobial activity

For determining antimicrobial activity, 2AT and type

strains of related Streptomyces species were grown in

starch casein nitrate broth at 28 �C on a rotary shaker

at 180 rpm. Fermentation was terminated on 4th day

and culture broths were centrifuged at 10,000 rpm for

30 min at 4 �C to separate the mycelium. Antimicro-

bial activity against various test organisms was

performed using agar well diffusion assay (Bauer

et al. 1996). The plates containing Mueller–Hinton

agar, yeast malt agar and potato dextrose agar were

seeded with test bacteria, yeasts and fungi, respec-

tively. Wells of 6 mm diameter were cut out from agar

plates, filled with supernatant (filtered through

0.22 lm filter, Pall Life Sciences) and inhibition

zones were recorded after 24–48 h of incubation.

Various test organisms used in the study included

Bacillus subtilis (MTCC 619), Mycobacterium

smegmatis (MTCC 6), Staphylococcus epidermidis

(MTCC 435), Escherichia coli (MTCC 1885),

Klebsiella pneumoniae sub sp. pneumoniae (MTCC

109), Enterobacter aerogenes (MTCC 111), Salmo-

nella typhi (MTCC 733), multi-drug resistant E. coli

(resistant to cephotaxime, ciprofloxacin, rifampicin

and cefoperazone), MRSA, VRE, Candida albicans

(MTCC 3017), Rhodotorula rubra (MTCC 248),

Colletotrichum acutatum (MTCC 1037), Cercospora

beticola (KJ461435), Fusarium oxysporum f.sp. dian-

thi (MTCC 6659), and Alternaria brassicicola (MTCC

2102).

Results and discussion

Isolate 2AT showed a range of chemotaxonomic and

morphological properties consistent with its classifi-

cation in the genus Streptomyces (Kampfer 2012).

Strain 2AT formed rectiflexibilis-type spore chains

with smooth spore surface ornamentation (Fig. 1,

Supplementary Fig. S1). The organism grew well on

most of the media, except ISP 5. No distinctive color

of substrate mycelium was recorded. The color of

aerial and substrate mycelia did not show much

variation on different media (Table 1). Strain 2AT did

not produce melanin pigment on tyrosine agar (ISP 7)

but produced blackish brown pigment on peptone

yeast iron agar (ISP 6). It hydrolyzed starch; but did

not hydrolyze casein, gelatin or urea. It degraded

esculin, did not produce indole from tryptophan and

gave negative results for MR-VP tests. It could neither

reduce nitrate nor utilize citrate. The strain produced

H2S and catalase and tolerated NaCl up to 2 %. Good

Fig. 1 Electron microscopic view of spores having smooth

surface

Antonie van Leeuwenhoek (2014) 105:943–949 945

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Table 1 Cultural characteristics of strain 2AT on various media

Characteristic SCNA Bennett’s

Agar

ISP 2 ISP 3 ISP 4 ISP 5 ISP 6 ISP 7

Growth Good Good Good Good Good Moderate Good Good

Sporulation Pinkish white White White Pinkish white Pinkish white White White Pinkish white

Colour of aerial

mycelium

Light yellow White Light grey Pinkish grey Pinkish white White Brown White

Colour of substrate

mycelium

White White White White Pinkish white White White White

Soluble pigment None None Blackish brown None None None None None

SCNA starch casein nitrate agar

Table 2 Phenotypic properties that distinguish strain 2AT from related Streptomyces species strains: 1, 2A; 2, S. flavotricini DSM

40152T; 3, S. toxytricini DSM 40178T; 4, S. globosus DSM 40815T

Characteristic 1 2 3 4

Aerial mycelium colour on

Yeast extract agar (ISP 2) Light yellow Light grey Absent Brown

Oatmeal agar (ISP 3) Light grey Pastel violet Light brown Beige

Inorganic salts–starch agar (ISP 4) Pinkish grey Pastel violet Greyish red Beige

Glycerol-asparagine agar (ISP 5) White None Yellowish pink Beige

Peptone-yeast extract iron agar (ISP 6) Brown None None None

Tyrosine agar (ISP 7) White Brown Beige Beige

Melanoid pigment - ? ? -

Production of diffusible pigment on ISP2 ? - ? ?

Geletin liquefication - ? ?/w ?/w

Citrate utilization - ? - -

Urease - ? - -

VP test - - ? -

Indole production - - - ?

H2S production ? - ? -

Nitrate reduction - w W -

Tolerance to NaCl 2 % 2 % 2 % 5 %

Utilization of

Arabinose - - ?/w ?/w

Sucrose ?/w ? - -

Xylose - - ? -/w

Inositol ?/w ? ? ?

Mannitol ?/w - - ?/w

Fructose ? - - ?

Rhamnose - ?/w - -

Raffinose - ? - ?

Cellulose ? ? - -

All four strains were positive for catalase, oxidase, starch hydrolysis and utilization of dextrose; negative for methyl red test and

casein hydrolysis

? Positive, - negative, w weak, -/w negative to weak, ?/w positive to weak

946 Antonie van Leeuwenhoek (2014) 105:943–949

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growth was recorded at temperature range of

15–37 �C and at pH range of 6.0–10.0. It utilized the

following sugars: D-glucose and fructose as sole

carbon sources, but did not utilize L-arabinose, xylose,

L-rhamnose, D-raffinose and cellulose (Table 2).

Strain 2AT had cell wall chemotype I which is

characteristic of the Streptomyces genus. The cell wall

contained LL-diaminopimelic acid and glycine.

Whole cell hydrolysates contained no characteristic

sugar. The menaquinones detected were MK-9(H6)

(58.2 %), MK-9(H8) (28.3 %) and MK-9(H10)

(13.5 %). The major phospholipids detected were

phosphatidylethanolamine, diphosphatidylglycerol,

phosphatidylglycerol (phosholipid type II). In addi-

tion, five unknown phospholipids and one unknown

glyco lipid were also present (Supplementary Fig. S2).

The major fatty acids found were anteiso-C15:0

(29.2 %), C16: 0 (16.0 %) and iso-C16: 0 (12.1 %).

The DNA G?C content of strain was estimated to be

70.5 ± 1.0 mol%.

Almost complete sequence (1463 bp) of 16S rRNA

gene of strain 2AT was determined (GenBank accession

no GQ906975) and compared with those of other closely

related taxa retrieved from the EzTaxon database.

Phylogenetic trees were constructed using neighbour-

joining (Fig. 2) and maximum-parsimony methods

(Supplementary Fig. S3). No significant differences

were observed in topologies generated by both neigh-

bour-joining and maximum-parsimony analyses. The

analyzed strain 2AT formed a distinct cluster in both

phylogenetic trees. However, low bootstrap values

(\50 %) do not reliably indicate the specific relatedness

between the species. The taxonomic integrity of the

isolate was also supported by the DNA–DNA related-

ness data as it shared DNA–DNA homology of

37.6 ± 0.6 %, 34.4 ± 0.5 % and 33.1 ± 0.4 % with

Table 3 Antimicrobial activity of strain 2AT and related Streptomyces species strains: 1, 2A; 2, S. flavotricini DSM 40152T; 3, S.

toxytricini DSM 40178T; 4, S. globosus DSM 40815T

Test organisms Reference number Inhibition zone (in mm)

1 2 3 4

Gram-positive bacteria

Bacillus subtilis MTCC 619 20 ± 0.1 - 11 ± 0.5 20 ± 0.1

Mycobacterium smegmatis MTCC 6 18 ± 0.5 - - 20 ± 0.5

Staphylococcus epidermidis MTCC 435 16 ± 0.5 - - 15 ± 0.5

Gram-negative bacteria

Escherichia coli MTCC1885 14 ± 0.5 - - -

Klebsiella pneumoniae sub sp. pneumoniae MTCC 109 10 ± 0.5 - - -

Enterobacter aerogenes MTCC 111 14 ± 0.5 - - -

Salmonella typhi MTCC 733 16 ± 0.5 - - -

Clinical isolates

MRSA 18 ± 0.1 - - 14 ± 0.5

E. coli 12 ± 0.5 - - -

VRE 11 ± 0.5 - - -

Yeasts

Candida albicans MTCC 3017 11 ± 0.5 - - -

Rhodotorula rubra MTCC 248 15 ± 0.5 -/w 12 ± 0.5 15 ± 0.5

Fungus

Colletotrichum acutatum MTCC 1037 12 ± 0.5 -/w 13 ± 0.5 ?/w

Cercospora beticola KJ461435 16 ± 0.5 -/w 12 ± 0.5 11 ± 0.5

Alternaria brassicicola MTCC 2102 16 ± 0.5 -/w 16 ± 0.5 ?/w

Fusarium oxysporum f.sp. dianthi MTCC 6659 11 ± 0.5 -/w 10 ± 0.0 10 ± 0.0

MTCC-Microbial Type Culture Collection, IMECH, Chandigarh (India)

? Positive, - negative, w weak, -/w negative to weak, ?/w positive to weak

Antonie van Leeuwenhoek (2014) 105:943–949 947

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the type strains, S. flavotricini DSM 40152T, S. globosus

DSM 40815T and S. toxytricini DSM 40178T, respec-

tively. These values are clearly well below the 70 % cut-

off point recommended for the assignment of strains to

the same genomic species (Wayne et al. 1987).

Strain 2AT demonstrated potent antimicrobial

activity against all the test microorganisms whereas

type strains of related Streptomyces species showed

weak activity (Table 3). The difference in antimicro-

bial activity further distinguishes 2AT from type

strains of related Streptomyces species.

Description of Streptomyces amritsarensis sp. nov

Streptomyces amritsarensis (am.rit.sar.en’sis. N.L.

masc. adj. amritsarensis, pertaining to Amritsar, a

holy city of Punjab state in India, where the type strain

was isolated).

Aerobic, Gram-positive, non-motile actinobacte-

rium which forms an extensively branched substrate

mycelium that bears aerial hyphae which carry

rectiflexibilis-type spore chains with smooth

surfaced spores (0.5 9 0.9 lm). Growth occurs from

15 to 37 �C, between pH 6–10 and can tolerate salt

concentration up to 2 % (w/v). Blackish brown

pigment is formed on peptone-yeast extract-iron

agar. Hydrolyses starch, but not urea or casein.

Gelatin is liquefied. Dextrose and fructose are used

as sole carbon sources for energy (at 1 %, w/v).

Additional phenotypic properties are cited in the text

and in Tables 1 and 2. Chemotaxonomic properties

are typical of the genus Streptomyces. Whole-cell

hydrolysate does not contain any characteristic sugar

and cell wall contains LL-diaminopimelic acid and

glycine. Major menaquinones are MK-9(H6), MK-

9(H8) and MK-9(H10). The phospholipid pattern is

PII type containing phosphatidylethanolamine (PE),

diphosphatidylglycerol (DPG), phosphatidylglycerol

(PG), five unknown phospholipids and one unknown

glyco lipid. The three most abundant fatty acids are

anteiso-C15: 0, C16: 0 and iso-C16: 0 and G?C content

of the genomic DNA of the type strain is

70.5 ± 1.0 mol%. Produces broad spectrum antimi-

crobial compounds active against various Gram-

S. sporoverrucosus NBRC 15458T (AB184684)

S .nojiriensis LMG 20094T (AJ781355)

S. xanthophaeus NBRC 12829T (AB184177)

S. vinaceus NBRC 13425T (AB184394)

S. cirratus NRRL B-3250T (AY999794)

S. lavendulae NBRC 12789T (AB184146)

S. spororaveus LMG 20313T (AJ781370)

S. colombiensis NRRL B-1990T (DQ026646)

S. avidinii NBRC 13429T (AB184395)

S. subrutilus DSM 40445T(X80825)

S. cinnamonensis NBRC 15873T (AB184707)

S. virginiae NBRC 12827T (AB184175)

S. toxytricini DSM 40178 T (AB184173)

S. globosus DSM 40815 T (AJ781330)

S. flavotricini DSM 40152 T (AB184132)

Strain 2AT (GQ906975)

S. katrae NBRC 13447T (AB184409)

S. racemochromogenes NRRL B-5430T (DQ026656)

S. polychromogenes NBRC 13072T (AB184292)

S. tanashiensis LMG 20274T (AJ781362)

S. nashvillensis NBRC 13064T (AB184286)

S. rubiginosohelvolus IFO 12912T (AY999864)40

100

100

60

64

46

94

46

98

99

87

80

62

6191

36

41

37

0.001

Fig. 2 Neighbour-joining tree based on 16S rRNA (1463 bp)

sequences, showing the phylogenetic relationship between strain

2AT and the related type strains obtained from the database of

type strains with validly published prokaryotic names at

EzTaxon-e identification service. Bootstrap values (expressed

as percentage of 1,000 replications) are given at the nodes

948 Antonie van Leeuwenhoek (2014) 105:943–949

123

positive and Gram-negative bacteria, drug resistant

bacteria and fungi.

The type strain, 2AT (=MTCC 11845T=JCM

19660T) was isolated from a soil sample collected

from Guru Nanak Dev University, Amritsar (India).

The species description is based on a single strain and

hence serves as a description of the type strain.

Acknowledgments The authors are thankful to Department of

Biotechnology, New Delhi, India for providing the financial

assistance.

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