Hindawi

International Journal of Microbiology

Volume 2020, Article ID 8817467, 8 pages
https://doi.org/10.1155/2020/8817467

Research Article
Molecular Identification and Antimicrobial Potential of
Streptomyces Species from Nepalese Soil

Karan Khadayat,1,2 Dawa Dindu Sherpa,2 Krishna Prakash Malla,2 Sunil Shrestha,2
Nabin Rana,2 Bishnu P. Marasini,2 Santosh Khanal,3 Binod Rayamajhee,3
Bibek Raj Bhattarai,1 and Niranjan Parajuli 1
1
Central Department of Chemistry, Tribhuvan University, Kirtipur, Kathmandu, Nepal
2
Department of Biotechnology, National College, Tribhuvan University, Naya Bazar, Kathmandu, Nepal
3
Department of Microbiology, National College, Tribhuvan University, Naya Bazar, Kathmandu, Nepal

Correspondence should be addressed to Niranjan Parajuli; [email protected]

Received 4 July 2020; Accepted 12 August 2020; Published 27 August 2020

Academic Editor: Sujata Prasad

Copyright © 2020 Karan Khadayat et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Streptomyces are widely used for the production of secondary metabolites with diverse biological activities, including antibiotics.
The necessity of alternative antimicrobial agents against multidrug-resistant pathogens is indispensable. However, the production
of new therapeutics is delayed in recent days. Thus, the isolation of new Streptomyces species has drawn attention. Nepal—a
country rich in biodiversity—has got high possibilities for the discovery of members of actinomycetes, especially in the higher
altitudes. However, in vain, only a few screening research works have been reported from Nepal to date. Streptomyces species were
isolated on ISP4 media, and characterization was performed according to morphological similarity and 16S rRNA sequence
similarity using bioinformatic tools. Ethyl acetate extracts of Streptomyces species were prepared, and the antimicrobial activity
was carried out using agar well diffusion technique. In this report, 18 Streptomyces species isolated from the soil were reported
based on sequence analysis of 16S rRNA. Among them, 12 isolates have shown antibacterial activity against extended-spectrum
beta-lactamase- (ESBL-) producing Escherichia coli. Here, we have also analyzed 16S rRNA in 27 Streptomyces species whose
whole-genome sequence is available, which has revealed that some species have multiple copies of the 16S gene (∼1.5 kb) with
significant variation in nucleotides. In contrast, some Streptomyces species shared identical DNA sequences in multiple copies of
16S rRNA. The sequencing of numerous copies of 16S rRNA is not necessary, and the molecular sequencing of this region is not
sufficient for the identification of bacterial species. The Streptomyces species-derived ethyl acetate extracts from Nepalese soil
demonstrate potential activity against ESBL-producing E. coli. Thus, they are potential candidates for antibiotics manufacturing in
the future.

1. Background Researchers have continuously explored novel, sustainable,

potent, and broad-spectrum bioactive compounds from
Multidrug-resistant pathogens have drawn global attention diverse sources, including Streptomyces species [6]. Strep-
as a significant challenge to treat and prevent the growing tomyces, the largest genus of class actinobacteria, is a Gram-
number of infectious diseases [1, 2]. Such resistance typically positive, spore-forming, filamentous, and aerobic bacterium
occurs as a result of drug inactivation, target alteration, and [7]. Streptomyces genome contains more than 20 gene
reduced accumulation unsettled to decreased permeability clusters to secondary metabolites of higher clinical impor-
and/or increased efflux [3]. Novel organisms with the ca- tance, including antibiotics that could tackle the rise of
pacity to produce new secondary metabolites or therapeutic antimicrobial resistance [8]. About two-thirds of natural
agents are immediately required to combat the fatal diseases antibiotics are isolated from actinomycetes, of which 70% is
caused by such antibiotic-resistant pathogens [4, 5]. produced from Streptomyces and accounts for 75% of
2 International Journal of Microbiology

clinically available antibiotics used in the treatment of sulfate, 18 gm Bacto agar, 1 gm sodium chloride, 2 gm
multiple infections in humans [9–12]. Many studies report ammonium sulfate, and 1000 mL distilled water at pH
that Streptomyces have tremendous potential to yield sec- 7.0 ± 0.1). The inoculum was then appropriately spread using
ondary metabolites, including anticancer drugs, antibiotics, a sterile glass spreader until the plates were dry and incu-
growth factors, and herbicides [13]. The higher percentage of bated at 28°C for 7–12 days [20].
GC content (∼70%) is present in Streptomyces spp. The 16S
rDNA analyses and DNA-DNA hybridization are significant
distinguishing properties that separate Streptomyces from 2.3. Characterization of Streptomyces. The characterization
other actinobacteria. Streptomyces from extreme or un- of Streptomyces was performed based on their Gram
touched habitats such as high altitude [14, 15] and desert staining, growth pattern, colony morphology, and the for-
[16, 17] are isolated and cultured to discover novel antibi- mation of soluble pigments, as suggested by Bergey’s Manual
otics. Extended-spectrum beta-lactamases (ESBLs) pro- of Systematic Bacteriology, Second Edition, Vol. 5, The
duced primarily by the bacteria Escherichia coli and Actinobacteria, Part A [21]. All of the isolates were grown on
Klebsiella pneumoniae confer resistance to most beta-lactam ISP4 media, and the morphology of each isolate was visually
antibiotics, including penicillins and cephalosporins. In- observed (such as colony characteristics, an earthy odor,
fections caused by these bacteria are being difficult to treat spore formation, and aerial and substrate mycelia of colo-
globally, and the mortality rate has sharply increased along nies). Sugar utilization tests and physiological tests such as
with prolonged hospital stay and greater economic burden motility, salt tolerance, and temperature tolerance were also
[18]. performed.
Due to the unique geographical niche of Nepal, soil
microbes, such as Streptomyces, have a high probability of 2.4. Genomic DNA Extraction and 16S rRNA Amplification of
producing novel secondary metabolites of diverse clinical Isolated Streptomyces Strains. Phenotypically identified iso-
value, which is much anticipated in the health care sector. In lates of Streptomyces were cultured on trypticase soy broth
this report, soil samples from 14 different environments of (TSB) using glass beads in 100 ml conical flasks for 3 days at
Nepal with varying from 86 m to 4,026 m above the sea level 28°C. After incubation, the supernatant part was discarded,
in altitude were collected for the isolation of Streptomyces and mycelia were harvested from the broth by centrifugation
species with antibacterial properties. This study is mainly at 4,000 rpm for 15 min. Genomic DNA was isolated by the
aimed at the screening of Streptomyces species that can standard phenol–chloroform method from the harvested
produce metabolites for the inhibition of ESBL-producing mycelia [22]. The universal primers 27 F:5′-AGAGTTT-
clinical isolate of E. coli, and their molecular identification GATCMTGGCTCAG-3′ and 1492 R:5′-ACGGY-
using 16S rRNA sequencing. Besides, 16S rRNA found in TACCTTGTTACGACTT-3′ were used for amplification of
other Streptomyces species reported in GenBank is also 16S rRNA from genomic DNA. The amplification was carried
reviewed for further scientific discourse. out in a 50 μL of total volume by using 125 ng of genomic
DNA as a template with 2X premix (Taq polymerase) and
2. Methods 10 μM of each primer. PCR conditions were maintained as
follows: initial 5 min for denaturation at 95°C, followed by 35
2.1. Collection and Pretreatment of Soil Sample. The soil cycles of 30s at 95°C, 30s at 51.4°C, and 120s at 72°C, and a
samples were collected from 14 different habitats of Nepal final extension of 10 min at 72°C (TAKARA Thermal Cycler,
(altitude from 86 m to 4,026 m above the sea level) such as Japan). The amplified products were examined by 0.8%
gardens, cultivated fields, barren land, open fields, organic agarose gel electrophoresis [23] stained by ethidium bromide.
manure pits, and soil from conserved forests, as shown in The purified PCR products were sequenced using the same
Supplementary Materials (Table S1). The samples were primers, 27 F and 1492R (Macrogen, Inc., South Korea).
collected digging around 5–10 cm depth from the surface of
the earth. Soil samples were then packed in sterile polythene
bags, labeled, and brought to the laboratory. They were air- 2.5. Sequence Analysis. The homology search of partial DNA
dried for 3-4 hrs at 45°C, crushed, and sieved before use for sequences of 16S rRNA was performed by comparing them
the isolation of Streptomyces species [19]. with the public database (NCBI) using the standard basic
local alignment search tool (BLAST) program. Multiple
alignments were conducted using Clustal [24], and the
2.2. Isolation of Streptomyces. One gram of soil was dissolved phylogenetic tree was made using MEGA version 6.0 by the
in 10 mL of sterile distilled water in the test tube and vor- neighbor-joining method with bootstrap values calculated
texed vigorously for 5 minutes. Then, the suspension was from 1,000 replications (Supplementary Materials,
heated at 80°C for 30 minutes and serially diluted up to 10−4 Figure S1) [25]. On the other hand, the multiple copies of
in the laminar hood; then 100 µL of each dilution was placed 16S rRNA present in the assembled genome sequencing of
on the International Streptomyces Project 4 (ISP4) agar the other 27 Streptomyces species given in the website (JGI
medium supplemented with nalidixic acid (20 mg/mL) and IMG Integrated Microbial Genomes and Microbiomes,
cycloheximide (50 mg/mL) (ISP4: 10 gm starch, 1 gm 2019) was evaluated through multiple DNA-DNA sequence
dipotassium phosphate (K2HPO4), 1 gm calcium carbonate, alignments using Clustal W (Supplementary Materials,
1 mg ferrous sulfate, 1 mg manganese chloride, 1 mg zinc Table S3).
International Journal of Microbiology 3

2.6. Extraction of Bioactive Compounds. The isolated 3.2. Antibacterial Assays. The antibacterial activity of ethyl
Streptomyces species were first cultured in the TSB and acetate extract (50 mg/mL) of isolates was evaluated against
incubated at 30°C for three days for the preparation of in- ESBL-producing clinical isolates of E. coli using well dif-
oculum. After full growth, 1% Streptomyces mycelium was fusion method. The results showed that crude extract
transferred aseptically into the fresh TSB and again incu- exhibited a zone of inhibition ranging from 10 to 13 mm
bated at 30°C for seven days. An equal volume of organic (size of wells: 6 mm) against ESBL-producing E. coli as
solvent, ethyl acetate, was used for extracting the bioactive compared to positive control neomycin with 19 mm zone of
compounds to an equal volume of culture. The mixture of inhibition, as shown in Figure 3 and Supplementary Ma-
culture with an organic solvent was shown in two layers terials, Table S2. Some pure isolates also showed antimi-
(organic layer contained secondary metabolites) and incu- crobial activity against S. aureus (ATCC 25923), E. coli
bated overnight in a rotary shaker. The mixture was then (ATCC 25922), K. pneumoniae (ATCC 700603), and Sal-
centrifuged, and the supernatant was taken. The concen- monella typhimurium (ATCC 14028) (data not included in
trated supernatant was further used for antimicrobial this report).
activity.

3.3. 16S rRNA Amplification. The PCR amplification of 16S

2.7. Antimicrobial Activity against ESBL-Producing E. coli. rRNA using a set of universal primers 27 F and 1492 R
The ESBL-producing clinical isolate of E. coli was already resulted in ∼1.5 kb product as compared to 1 kb ladder (New
confirmed by coauthors in the previous study [26]. Muel- England Biolabs) for all isolates. The amplified products
ler–Hinton agar (MHA) plates were spread with test or-
ganisms (0.5 McFarland turbidity standard) using a sterile
were then purified with DNA Clean and Concentrator -5
(Catalog no. D4003), Zymo Research (USA), following the
™
cotton swab. MHA plates were bored with 6 mm-diameter manufacturer’s instructions, and the DNA sequencing using
sterile cork borer, and 30 μL ethyl acetate extract was loaded universal primers was performed in Macrogen Inc., South
into wells. The plates were then incubated at 37°C for 24 h, Korea.
and the zone of inhibition was measured [27]. To validate the
experiment, negative control (ethyl acetate) and positive
control (1 mg/mL neomycin) were also maintained, and each 3.4. Molecular Characterization. The proximity of the 16S
experiment was triplicated. We had also examined the an- rRNA gene of the isolates was compared with the sequences
timicrobial activity of some isolates with Staphylococcus available in public databases (GenBank, EMBL, and DDBJ)
aureus (ATCC 25923), E. coli (ATCC 25922), and using the Nucleotide Basic Local Alignment Search
K. pneumoniae (ATCC 700603) by the perpendicular Tool—BLASTN version 2.2.29 [28] Sequence homology was
streaking method as primary screening. compared with 16S rRNA gene sequences available in the
database using the FASTA algorithm. The 16S rRNA gene
3. Results sequences of other Streptomyces, representing the type
strains of Streptomyces species, were retrieved from the
3.1. Characterization of Streptomyces. Soil samples collected GenBank. The sequence was aligned using Clustal W ver.
from various environments varied in texture, and 18 2.01, and the phylogenetic tree was constructed using MEGA
Streptomyces isolates were successfully isolated (Table S1). ver. 6 by the neighbor-joining method (Figure S1). The
Among them, no isolate was isolated from the soil of assigned GenBank DNA sequences DA-1, DA-2, DA-3, DA-
Rasuwa, two isolates from Thali, Dhangadi, Chitlang, 4, KM-1, KM-2, KM-3, KM-5, KM-6, KM-8, SA1, SA2, SA3,
Tapoban, and Taudaha soil and one isolate from each SA4, SA4, SA5, SA6, SA7, and SA8. The accession numbers
remaining eight types of soil. The isolates were identified are mentioned in Table 1, and phylogenetic analysis revealed
as Streptomyces based on their mycelial and cellular that 18 isolates shared some evolutionary similarities
morphology observed under a microscope and no other (Supplementary Materials, Figure S1).
species such as Nocardia spp. and Micromonospora spp.
were obtained. All these isolates attained maximum
growth after seven days of incubation with colored 3.5. Genome-Wide Analysis of 16S rRNA. The multiple copies
sporulation ranging from dark grey, grey, dark brown, of 16S rRNA are found in 27 Streptomyces species in their
brownish, whitish, and yellowish-white, as indicated in whole-genome sequences. In 7 species, DNA sequences in
Figure 1. The microscopic examination of actinomycete multiple copies of 16S rRNA are highly conserved, but in 20
strains showed Gram-positive with hair-like mycelium, as others, there is little variation and per-site gaps in the aligned
shown in Figure 2. The physiological test showed that the predicted by multiple sequences. Streptomyces violaceusniger
isolates were nonmotile, unable to produce hydrogen Tu 4113 has only one 16S rRNA in the genome. The nu-
sulfide, optimum growth between 28°C and 37°C, and able merous copy of 16S rRNA present in the assembled genome
to tolerate up to 5% NaCl. In contrast, the sugar utilization sequencing of Streptomyces is given in Supplementary
test revealed that most of the strains under study were able Materials, Table S3. Our analysis revealed that DNA se-
to utilize glucose, arabinose, sucrose, xylose, and fructose quencing of a single copy of 16S rRNA in the genome could
but were not able to utilize mannitol, inositol, raffinose, be sufficient for the identification of genus Streptomyces
and rhamnose. from class actinomycetes.
4 International Journal of Microbiology

Figure 1: Colony morphology indicating aerial (a) and substrate (b) mycelia of isolated Streptomyces species.

(a) (b)

(c) (d)

Figure 2: Gram staining of isolated actinomycetes under 100x magnification.

International Journal of Microbiology 5

(a) (b)

(c) (d)

Figure 3: Antibacterial activity against ESBL-producing E. coli shown by crude extract of Streptomyces species.

4. Discussion to Gram-positive bacteria [32]. Hence, actinomycetes that

produce secondary metabolites against Gram-negative might
The present study outlines molecular phylogeny, and anti- play a significant role against antibiotic-resistant bacteria,
microbial activity of Streptomyces isolated from Nepalese such as ESBL-producing E. coli. Previous studies suggested
soil. Till now, more than 7,000 compounds produced by that ethyl acetate extract of actinomycetes, isolated from
Streptomyces species have been identified [6]. Continuous Nepalese soil of different altitudes, can produce secondary
screening of Streptomyces will lead to the discovery of metabolites, which showed antimicrobial activity against
numbers of new compounds with diverse applications [29]. different ATCC strains [33–36].
Thus, the reported 18 Streptomyces species from different To our knowledge, the antimicrobial activity against
environmental niches of Nepal would be beneficial for future ESBL-producing E. coli using secondary metabolites of
antibiotics discovery program. Streptomyces and their molecular characterization have not
In this study, the ethyl acetate extracts showed antimi- been reported earlier from Nepal.
crobial activity against ESBL-producing E. coli. Previously, From the analysis of partial 16S rRNA sequences de-
Ramachandran et al. have also suggested that ethyl acetate posited in GenBank (Table 1), it was revealed that all isolates
extract of actinomycetes has antibacterial activity against belong to a species of the genus Streptomyces with 80–99%
ESBL-producing E. coli [30], and this solvent is considered similarity with the 16S rRNA sequence of its closely related
better for the extraction of active metabolites from actino- species. Bacterial genetic marker, 16S rRNA, often exists as a
mycetes [31]. In several reports, secondary metabolites from multigene family or operons in chromosomal DNA. The
actinomycetes have shown the higher inhibition against function of this gene over time has not been changed, and
Gram-negative bacteria; among them, E. coli is more sus- the size of this gene is large enough for the informatics
ceptible. However, due to the presence of double-membrane purposes [37]. The complete gene sequence of 16S rRNA
barrier and transmembrane efflux, Gram-negative bacteria consists of approximately 1,500 bp with significantly con-
are more resistant to antimicrobial compounds as compared served regions and nine variable regions that allow broad
6 International Journal of Microbiology

Table 1: 16S rRNA sequence analysis of Streptomyces species and potent secondary metabolites.
Match in the databases
Sample no. Accession number
Species Identity (%) Identifier
Streptomyces spp. ZG731 96 GQ985455.1
SA1 LC427859
Streptomyces coelicoflavus strain HQA809 93 KT758401.2
Streptomyces spp. BCG69 80 KF956734.1
SA2 LC425654
Streptomyces spp. 219839 80 HQ992728.1
Streptomyces spp. strain D3 97 KX762051.1
SA3 LC427860
Streptomyces lividans strain jx-02 97 KC898819.1
Streptomyces spp. strain D3 98 KX762051.1
SA4 LC427861
Streptomyces lividans strain YLA0 98 KT362142.1
Streptomyces spp. CMU-AC2 96 LC073311.1
SA5 LC427862
Streptomyces spp. CMU-AB225 96 LC073310.1
Streptomyces spp. JSM 147611 96 KR817740.1
SA6 LC427863
Streptomyces violascens strain G8A-22 96 HQ238389.1
Streptomyces spp. DHS C014 97 KP986577.1
SA7 LC427864
Streptomyces tumenensis 97 AM180560.1
Uncultured Streptomyces spp. clone T1S-05 82 GQ369231.1
SA8 LC427865
Streptomyces ryensis strain zw24 83 MH337934.1
Streptomyces spp. 34005 98 GU263848.1
KM1 MT463712
Streptomyces strain P14S1 98 KX673842.1
Streptomyces globisporus subsp. globisporus isolate XSD-114 85.1 EU273549.1
KM2 MT463715
Streptomyces cavourensis strain TSM 11 85 MK789724.1
Streptomyces pratensis strain EA5 93.2 KU973961.1
KM3 MT463732
Streptomyces spp. QLS12 93.5 KU973961.1
Streptomyces coelicoflavus strain 3-2 98.3 KJ571034.1
KM5 MT464457
Streptomyces coelicoflavus strain HQA020 98.1 KT758352.1
Streptomyces spp. 193322 96.2 KU982617.1
KM6 MT464460
Streptomyces parvus strain T23 96.2 KU317906.1
Streptomyces canus strain IMCC 34906 97.4 MK138629.1
KM8 MT464462
Streptomyces spp. strain ST228 97.4 KX906839.1
Streptomyces globisporus strain AHS10 99 KU981096.1
DA1 MT441544
Streptomyces violascens strain EA27 98.2 KU973982.1
Streptomyces naganishii NRRLB-1816 98.4 NR_043831.1
DA2 MT459245
Streptomyces spp. FXJ1.447 98.4 KP126357.1
Streptomyces pratensis 190525 97.1 KU973967.1
DA3 MT459275
Streptomyces spp. strain WA22-1-2 97 KY206810.1
Streptomyces spp. strain HN25 98.7 MF397913.1
DA4 MT463685
Streptomyces diastaticus subsp. ardesiacus strain AR-39 98.7 KX055839.1

taxonomic spectrum and taxonomic discrimination, re- copies of 16S rRNA analysis [44]. Ten Streptomyces strains
spectively. However, there is some discrepancy regarding the isolated from three different lichens were found to have
molecular identification of bacterial species based on 16S similar 16S rRNA gene sequences [45, 46]. The analysis of
rRNA. Guo et al. have claimed that sequencing of 16S rRNA multiple copies of 16S rRNA in different Nocardia strains
is not useful for closely related strains and only limited to the suggests that only BLAST analysis could not confirm the
distinction of slightly related Streptomycetes [38]. species present in the isolates [47]. Two copies of 16S rRNA
Some studies have reported that multiple copies of 16S genes contained in actinomycete, Thermobispora bispora,
rRNA are limited and have less probability of affecting the differ in nucleotides sequence at 98 positions (6.4% of
phylogenetic analysis of the species because, in most cases, the complete nucleotides sequence) having six regions of in-
sequence of these multiple copies is entirely or nearly identical sertion and deletions [48]. A study suggested transitional
[39–41]. However, others have shown that with an increase in and transversional substitutions as two distinct mechanisms
the number of multiple copies of 16S rRNA, the variation in for the occurrence of multiple copies of 16S rRNA in 33
sequence also increases [42], and the 16S rRNA sequence is Streptomyces strains (6.9%) out of 475 isolates [49]. Thus, the
not enough criteria to assure species identity [43]. sequencing of multiple copies of 16S rRNA seems to be not
In silico analysis of 16S rRNA of 174 actinobacteria useful for the identification of species.
showed the phylum contains 3.402 ± 1.720 average copies of
16S rRNA, of which 1.569 ± 0.869 copies are not 100% 5. Conclusion
similar to other copies. The results infer that the average
maximum variation is 1.121 ± 2.400, along with an average The present study demonstrates that Streptomyces species
minimum similarity of 99.927 ± 0.158 based on multiple isolated from different altitudes of Nepal showed potential
International Journal of Microbiology 7

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The authors would like to thank Mr. Raju Shreastha for Microbiology, vol. 2019, pp. 1–20, 2019.
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