Microbial Pathogenesis 135 (2019) 103624

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Microbial Pathogenesis
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Bacterial endophytes from Artemisia nilagirica (Clarke) Pamp., with T

antibacterial efficacy against human pathogens
A. Ashithaa, S.J. Midhuna, M.A. Sunila, T.U. Nithina, E.K. Radhakrishnana, Jyothis Mathewa,*
a
School of Biosciences, Mahatma Gandhi University, Priyadarshini Hills P.O, Kottayam, 686560, Kerala, India

A R T I C LE I N FO A B S T R A C T

Keywords: A study was conducted to isolate and characterize endophytes from Artemisia nilagirica, a traditional medicinal
Artemisia nilagirica plant. The plant was collected from Western Ghats, India. Endophytes isolated included Arthrobacter sp. WWAT1,
Endophytes Pseudomonas sp. WYAT2, Microbacterium sp. WYAT3, Psychrobacter sp. WBAT4, Enterobacter sp. WWAT5, Bacillus
Chromobacterium violaceum sp. WBAT6, Kosakonia cowanii WBAT7, Bacillus sp. WBAT8, Bacillus sp. WBAT9, Chromobacterium violaceum
Burkholderia sp.
WVAT6, Serratia sp.WPAT8 and Burkholderia sp. WYAT7. Of these two bacteria, Chromobacterium violaceum
Antibacterial
Western Ghats
strain WVAT6 and Burkholderia sp. strain WYAT exhibited antibacterial property against human pathogens.
Similar to the environmental isolates, Burkholderia sp. WYAT7 showed pleomorphism and produced different
enzymes, whereas like clinical strains they showed multidrug resistance, for their survival in different en-
vironmental conditions. Chromobacterium violaceum WVAT6 exhibited rod shape morphology and showed
multiple drug resistance except to erythromycin, tetracycline and gentamicin antibiotics. Both produced biofilm
and enzymes such as protease and lipase. The antimicrobial compounds from these endophytes may find ap-
plication in the preparation of antimicrobial formulations.

1. Introduction
Endophytes, which include bacteria and fungi, live within the
healthy tissues of living plants and are essential components of plant
micro-bionetwork. Endophytes have been established in every plant
species examined to date and are ubiquitous with rich biodiversity [1].
The host plant and some co-existing endophytes, after the extensive
phase of evolution, have created a unique relationship with one an-
other, which can even influence the quality and quantity of metabolic
products in plants. The host plant genome evolution is affected by its
endophytic community [2,3] and also the unique chemical environ-
ment of host plant influences the metabolite profile of endophytes. The
close interactions between the plant and bacterial endophytes lead to
the synthesis of broad range of potential secondary metabolites by
bacterial endophytes [4]. It is possible that the beneficial characteristics
of medicinal plants are due to the metabolites produced by their en-
dophytic community [5,6]. Recently many researchers and pharma-
ceutical companies are using ethnobotanical approach to come up with
novel drugs from endophytes.
Various researchers have reported the isolation of endophytic bac-
teria from different plants existing in diverse ecosystems [7]. The en-
dophytes present in the plant may either restricted at the point of entry
or inhabit within the cells, the vascular system, the intercellular spaces
or can be extended all over the plant [8]. Bacterial endophytes have
been isolated from leaves, roots, stem, seeds, fruits and as well as from
tubers of different plants [9]. Optimization of isolation and purification
steps is important while performing endophyte isolation. The isolation
procedure should be sensitive enough to recover endophytes and also
necessarily eliminate epiphytes from the surface of plants [10]. The
bacteria enter the plant tissue during evolution and establishes an en-
dophytic relationship [11]. The endophyte entry and attachment to
plant tissues are either aided with the synthesis of cell wall hydrolyzing
enzymes or by developing other mechanisms such as biofilm production
[8,12]. The genetic and environmental factors also affect bacterial ad-
hesion, plant colonization and plant-bacterial interactions [13]. The
endophytes of medicinal plants have been reported as rich sources of
antimicrobial, antioxidant, anti-cancer, anti-inflammatory and im-
munosuppressive agents [14,15]. Antimicrobial compounds from bac-
terial endophytes were isolated from Chinese medicinal plants and they
also acted as biocontrol agents [16].
The traditional pharmacopoeias of different ethnic groups have
described about several plant species of the genus Artemisia. Among
these, Artemisia nilagirica is extensively used in Ayurvedic and
Homeopathic medicine. Artemisia nilagirica is usually found in hilly
areas of India and called as Indian wormwood. This medicinal plant is
traditionally used for curing various skin diseases, nervous disorders,

*
Corresponding author.
E-mail address: prof.dr.jyothismathew@gmail.com (J. Mathew).

https://doi.org/10.1016/j.micpath.2019.103624
Received 9 April 2019; Received in revised form 3 June 2019; Accepted 16 July 2019
Available online 16 July 2019
0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
A. Ashitha, et al. Microbial Pathogenesis 135 (2019) 103624

Fig. 1. Phylogenetic tree analysis of 16S rDNA sequences of bacterial isolates along with reference sequences from NCBI. The evolutionary history was analyzed using
the Neighbor-joining method. The percentage of replicate tree in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to
the branches.

epilepsy, and inflammation. Based on the previous works of literature,
it was reported that oil and leaf extract had antimicrobial, antifungal,
larvicidal and insecticidal activity [17]. Artemisia nilagirica is also used
for cancer treatment. To the best of our knowledge to date, no studies
on its bacterial endophytes have been reported. In this context, aim of
this work was to isolate and identify endophytic bacteria from the plant
Artemisia nilagirica (Clarke) Pamp. and to characterize them by studying
morphological properties, biofilm production, susceptibility to anti-
biotics and antibacterial properties.
2. Materials and methods
2.1. Plant material and study site
The medicinal plant Artemisia nilagirica (Clarke) Pamp. was col-
lected from their natural habitat in Wayanad regions (11.6854° N,
76.1320° E), a part of Western Ghats, Kerala, India. The whole plant
was collected from different regions of Wayanad district.

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A. Ashitha, et al. Microbial Pathogenesis 135 (2019) 103624

Table 1
Summarized result of BLAST and the accession numbers of isolates.
Sl.No. Assigned code and accession number of submitted Closest NCBI match with accession number Percentage similarity Identification of isolates
sequences

1 WWAT1; MH680713 Arthrobacter sp. strain G8; HQ111068.1 97% Arthrobacter sp..
2 WYAT2; MH680714 Pseudomonas aeruginosa strain MCCB; EF062514.2 98% Pseudomonas sp.
3 WYAT3; MH680715 Microbacterium oxydans strain 2345; EU714339.1 98% Microbacterium sp.
4 WBAT4; MH680716 Psychrobacter maritimus strain 0108; KP236243.1 96% Psychrobacter sp.
5 WWAT5; MH680717 Enterobacter hormaechei strain D15; KJ863539.1 95% Enterobacter sp.
6 WBAT6; MH680718 Bacillus sp. strain WR-2; KU159243.1 97% Bacillus sp.
7 WBAT7; MH680719 Kosakonia cowanii; LC040935.1 100% Kosakonia cowanii
8 WBAT8; MH680720 Bacillus subtilis strain BA14; KU510073.1 95% Bacillus sp.
9 WBAT9; MH680721 Bacillus aryabhattai strain L54; KU179348.1 95% Bacillus sp.
10 WVAT6; MG787949 Chromobacterium violaceum strain BF-R1; KY292417.1 100% Chromobacterium violaceum
11 WPAT8; MF535458 Serratia marcescens strain DAP33; EU302858 98% Serratia sp.
12 WYAT7; MF535457 Burkholderia sp. strain C25; MF784500.1 96% Burkholderia sp.

Table 2 Tgg CTC-3′) and 1525R (5′-AAg gAg gTg WTC CAR CC-3′). The 16S
Isolation of endophyte bacteria from Artemisia nilagirica (Clarke) Pamp. rDNA sequence data was subjected to BLAST analysis. For the phylo-
Bacterial isolates Number of isolates from different plant parts Total genetic analysis, the similar sequences were retrieved from NCBI using
BLAST. The selected sequences were first aligned with the ClustalW
Leaf Stem Root program using Bioedit software. The aligned data were used for further
phylogenetic analysis using MEGA7 [18].
Arthrobacter sp. 2 1 – 3
Pseudomonas aeruginosa 4 5 4 13
Microbacterium oxydans 4 2 – 6 2.5. Screening for antagonistic activity of endophytic bacteria against potent
Psychrobacter maritimus 2 – – 2
bacterial pathogens
Enterobacter hormaechei 2 – – 2
Bacillus sp. – 5 6 11
Kosakonia cowanii 2 2 – 4 Preliminary antibacterial study of isolates was done by perpendi-
Bacillus subtilis – – 5 5 cular streak method on Mueller- Hinton agar (MHA) plates against
Bacillus aryabhattai – 3 3 6 potent human pathogens. The bacterial pathogens such as Salmonella
Chromobacterium violaceum – 3 – 3
typhi (MTCC 733), Salmonella paratyphi, Staphylococcus aureus (MTCC
Serratia marcescens – 8 6 14
Burkholderia sp. 2 4 – 4 1430), Pseudomonas aeruginosa (MTCC 2453), Klebsiella pneumoniae
Total endophytes isolated 18 33 24 75 (MTCC 432) and Escherichia coli (MTCC 1610) were used in this study.
The endophytic bacteria were streaked in the center of the nutrient agar
plate as a straight line and the pathogens as perpendicular to the en-
2.2. Surface sterilization of plant materials dophyte [19]. The plates were then incubated for 24 h at 30 ± 4 °C and
observed for growth inhibition.
Endophytic isolation was carried out under aseptic conditions.
Samples were washed gently in running tap water to remove dust and
2.6. Antibacterial activity of culture extracts of selected isolates against
debris. Different parts of plants such as stem, leaves and root were first
potent bacterial pathogens
surface sterilized by 3% of sodium hypochlorite (NaOCl) (4% w/v
available chlorine). The different plant parts were treated with 75%
The cellular preparations were made according to the modified
ethanol for 1min followed by immersion in 1% NaOCl (4% w/v avail-
method of Beiranvand et al. (2017) [20] . The two selected isolates
able chlorine) then in 75% ethanol for 30 s. The plant materials were
were cultured in nutrient broth and incubated for 14 days at 30 ± 4 °C.
finally rinsed with deionized sterile distilled water to remove sterilants
After that, the cultures were extracted using three methods. The culture
and blot dried on sterile tissue paper.
broth was separated to three equal parts for the preparation of three
types of extracts. For the preparation of ethyl acetate extract of culture
2.3. Isolation of endophytes EAE(C), one part of the culture broth was mixed with the equal amount
of ethyl acetate in a separating flask. The organic supernatant was se-
Proper sterilized plant materials, i.e. the stems and root were cut parated and centrifuged at 8000 rpm for 10min. The ethyl acetate su-
into 0.75 ± 0.25 cm pieces and the leaves into 4 mm x 0.5–1 cm pieces pernatant was transferred into a clean flask and dried at 50 °C. The
and were placed on nutrient agar plates. To ensure the absence of dried extract was dissolved in 2 ml DMSO. For the preparation of ethyl
epiphytic bacteria, the finally rinsed water was plated on to nutrient acetate extract of boiled and cooled culture EAE (BC), another part of
agar plates. Plates were then incubated at room temperature the media was incubated in boiling water for 5 min followed by cold
(30 ± 4 °C). After 5 days of incubation bacteria grown were properly water for 5 min it was then mixed with ethyl acetate in the ratio of 1:1.
subcultured and pure cultures were maintained at 4 °C. Then the samples were processed according to the first method. For the
preparation of ethyl acetate extract of the sonicated culture EAE (SC),
2.4. Identification of the isolates the culture was sonicated for 3 min at 130W and subsequent extraction
was done as described in the first method. The antibacterial activity of
The molecular identification of isolates was done by 16S rDNA se- three extracts were tested by well diffusion method against pathogenic
quencing method. The isolates were inoculated into 20 ml of Luria bacteria such as Salmonella typhi (MTCC 733), Staphylococcus aureus
Bertani's (LB) broth in a 100 ml conical flask and incubated for 5 days at (MTCC 1430), Pseudomonas aeruginosa (MTCC 2453), Klebsiella pneu-
room temperature. For DNA isolation, the cells were collected by cen- moniae (MTCC 432), Escherichia coli (MTCC 1610), Salmonella paratyphi,
trifugation and the pellet was used as per protocols in DNA extraction Bacillus subtilis and Acinetobacter baumannii. The zone of inhibition was
Kit (Sigma Aldrich, USA). The 16S rDNA was amplified by Mycycler™ measured for each bacterial pathogen and wells containing 20 μl
(Bio-Rad, USA), using the universal primers 27F (5′-AgA gTT TgA TCM streptomycin (1 mg/ml) served as control.

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A. Ashitha, et al. Microbial Pathogenesis 135 (2019) 103624

Fig. 2. Antagonistic activities of endophytic bacteria against pathogens. Antagonistic efficacy of all the twelve isolates was examined against human bacterial
pathogens like Staphylococcus aureus (MTCC 1430), Salmonella paratyphi, Salmonella typhi (MTCC 733), Pseudomonas areuginosa (MTCC 2453), Klebseiella pneumonia
(MTCC 432), and Escherichia coli (MTCC 1610). A- Arthrobacter sp. WWAT1, B- Pseudomonas sp. WYAT2, C- Microbacterium sp WYAT3, D-. Psychrobacter sp. WBAT4,
E- Enterobacter sp. WWAT5, F- Bacillus sp. WBAT6, G- Kosakonia cowanii WBAT7, H- Bacillus sp. WBAT8,I- Bacillus sp. WBAT9, J- Chromobacterium violaceum WVAT6,
K- Serratia sp. WPAT8, L- Burkholderia sp.WYAT7. J and L showed better activity against most of the pathogens.

Table 3
Antibacterial activity of culture extracts of selected endophytic bacteria against human pathogens.a
Extracts Zone of inhibition against different bacterial pathogens (mm)

A. baumannii B.subtilis E.coli K.pneumoniae S.aureus P.aeruginosa S.typhi S.paratyphi

Burkholderia.sp EAE(C) 16 ± 0.57* 13.33 ± 11.66 ± 0.33 10.33 ± 0.33 10.33 ± 0.33 11 0 0
0.33*
Burkholderia.sp 16* 12.33 ± 0.33 12.66 ± 0.33* 11 11.66 ± 0.33 11 0 0
EAE(BC)
Burkholderia.sp 18 ± 0.57** 14.33 ± 0.33* 13.33 ± 0.33* 11.66 ± 0.33* 16.33 ± 0.33* 12.33 ± 0.33 0 0
EAE(SC)
C.violaceum EAE(C) 14.33 ± 0.33* 11 11 10.33 ± 0.33 12.66 ± 0.33* 11 0 0
C.violaceum EAE(BC) 14.66 ± 0.33* 12.66 ± 0.33 11.33 ± 0.33 11 13* 12.66 ± 0.33 0 0
C.violaceum EAE(SC) 15* 14* 11.66 ± 0.33 11.33 ± 0.33 13.66 ± 0.33* 13* 0 0
Control (Streptomycin) 26.66 ± 20*** 23.33 ± 16.66 ± 19.33 ± 23*** 23*** 22.33 ± 0.33***
0.33*** 0.33*** 0.33*** 0.33***

a
Data are represented as mean ± SE (n=3). Values with same superscript symbols are not statistically different. Significance level * < ** < ***.

2.7. Scanning electron microscopic (SEM) analysis of two selected isolates 2.8. Screening for enzyme production

SEM was carried out to examine morphological features of the two
isolates Chromobacterium violaceum WVAT6 and Burkholderia
sp.WYAT7. The cultures were grown overnight in nutrient broth and
cells were collected by centrifugation at 5000 rpm for 5 min and were
resuspended in PBS solution. The samples were then fixed with 2.5%
(W/V) glutaraldehyde for 2 h and washed with distilled water and then
dehydrated with ethanol at increasing concentrations of 20%, 40%,
60% and 100% for 2 min each. Fixed and dehydrated smears were
coated with gold and evaluated by JEOL- JSM- 6390 model SEM in-
strument.
2.8.1. Protease production
The bacteria were streaked on to skim milk agar plates and in-
cubated at 30 ± 4 °C for 24–48 h and examined for formation of clear
zone around the colonies [21].
2.8.2. Screening for lipase production
Lipolytic activity of the bacterial strains was tested using tributyrin
agar plate assay method (TBA) [22]. Tributyrin agar media were ster-
ilized and poured into petri plates. The bacteria were streaked on the
tributyrin agar plates and was incubated at 30 ± 4 °C for 24 h to ob-
serve zone.

4
A. Ashitha, et al.
Fig. 3. SEM images of selected isolates. The morphological characteristics such as size and shape of the isolates are evident in SEM images. a-illustrate the rod shape
of the bacteria Chromobacterium violaceum WVAT6, b-shows the coccobacillius shape of Burkholderia sp. WYAT7.
2.9. Screening for biofilm production by Congo red agar method
Congo Red Agar method is a simple qualitative method to detect
biofilm production [23]. Congo red stain was prepared and autoclaved
separately from other medium constituents. It was then mixed with
autoclaved brain heart infusion agar (55 °C) containing sucrose. CRA
plates were inoculated with test organisms and incubated for 24 h at
30 ± 4 °C.
2.10. Antibiotic sensitivity test of selected endophytes
Kirby- Bauer disc diffusion method was employed to determine the
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Microbial Pathogenesis 135 (2019) 103624
Fig. 4. Screening for enzyme production. a-showing
protease production by Burkholderia sp. WYAT7 on
skim milk agar plates with large zone of clearance,
b-protease production by Chromobacterium viola-
ceum WVAT6 on skim milk agar plates with large
zone of clearance, c-lipase production by
Burkholderia sp. WYAT7 on tributyrin agar plates
with slight clearance around the colony, D-lipase
production of Chromobacterium violaceum WVAT6
on tributyrin agar plates with slight clearance
around the colony.
antibiotic sensitivity or resistance of the selected strains against various
antibiotics available. The overnight grown cultures of selected bacterial
strains were swabbed on to Muller- Hinton agar plates and different
antibiotic discs were placed on them. The antibiotic discs used were
Amoxicillin (30mcg), Gentamicin (30mcg), Erythromycin (10mcg),
Penicillin- G (2 units), Vancomycin (10mcg) and Tetracycline
(10mcg).The plates were incubated for 24 h at 30 ± 4 °C and examined
for the zone of clearance.
2.11. Statistical analysis
The differences in the zone of inhibition produced by three fractions
A. Ashitha, et al.
Fig. 5. Biofilm production in CRA medium. a – showing biofilm production by
Chromobacterium violaceum WVAT6, b-dry crystalline black colony produced by
Burkholderia sp.WYAT7, c-non biofilm producer, Aeromonas cavae will not
produce black colony in CRA medium.
of two selected isolates against human bacterial pathogens were as-
sessed separately with the zone of positive control (streptomycin). It
was done by one way ANOVA, followed by Tukey's test at the level
p ≤ 0.05. The statistical analysis was performed using the GraphPad
Prism© version 5.03 for Windows (GraphPad Software, San Diego, CA,
USA).
3. Results
3.1. Isolation and identification of endophytic bacteria from Artemisia
nilagirica (Clarke) pamp
The healthy plants of Artemisia nilagirica (Clarke) Pamp. were col-
lected from Wayanad district of Kerala, India. The specimen sample was
deposited in the University of Calicut Herbarium with Accession
number 6855. The plant was identified by Prof. Dr. Pradeep A.K
(Taxonomist), Department of Botany, University of Calicut, Kerala.
The isolation of endophytic bacteria from the medicinal plant
Artemisia nilagirica from distinct regions of Wayanad resulted in ob-
taining 75 isolates. The effectiveness of surface sterilization of plant
parts of Artemisia nilagirica was ensured as there was no microbial
growth observed in the nutrient agar plate poured with the finally
rinsed water and incubated for 5 days. Based on the visible morpho-
logical differences and Gram staining, similar endophytes were sub-
jected to molecular identification by 16S r DNA sequencing. This re-
vealed the isolates as Arthrobacter sp., Pseudomonas sp., Microbacterium
sp., Psychrobacter sp., Enterobacter sp., Bacillus sp., Kosakonia cowanii,
Table 4
Antibiotic susceptibility of isolates.
Isolates Zone of inhibition surrounding the antibiotic discsa
Amoxicillin 30mcg Gentamicin 30mcg
(AMX) (GEN)
Burkholderia sp. strain WYAT7 0(R) 15 mm(IM)
Chromobacterium violaceum strain 0(R) 24 mm(S)
WVAT6
a
R-resistant, IM-intermediate, S- susceptible.
Microbial Pathogenesis 135 (2019) 103624
Chromobacterium violaceum, Serratia sp. and Burkholderia sp.The phy-
logenetic analysis was done using Bioedit and MEGA7 software, and the
evolutionary tree was constructed by the neighbor-hood joining method
(Fig. 1). All the isolates got accession number from Gene Bank. The
results are summarized in Table 1.
The results demonstrated that the population density of endophytes
in medicinal plant Artemisia nilagirica was higher in the stem portion
than in the roots and leaves (Table .2). The isolate Pseudomonas sp. was
present in all plant parts and the isolates except Psychrobacter sp., En-
terobacter sp. and Bacillus sp. strain were present in the stem. The ma-
jority of the bacterial endophytes isolated were pigment producers.
3.2. Screening for antagonistic activity of endophytic bacteria against
bacterial pathogens
Among the isolates tested, Chromobacterium violaceum WVAT6 and
Burkholderia sp. WYAT7 showed antagonistic activity against most of
the bacterial pathogens. The lowest antagonistic activity was showed by
Bacillus sp. WBAT6 and Bacillus sp. WBAT9. The results are shown in
Fig. 2. Further studies were carried out using Chromobacterium viola-
ceum WVAT6 and Burkholderia sp.WYAT7.
3.3. Antibacterial activity of culture extracts of selected isolates against
potent bacterial pathogens
The three preparations from Burkholderia sp. WYAT7 culture
showed antibacterial activity against all the bacterial pathogens except
Salmonella typhi and Salmonella paratyphi. These three extracts showed
better zone of inhibition for Acinetobacter baumannii, when compared
with the other pathogens. In that, EAE (SC) preparation showed highest
inhibition with 18 ± 0.57 mm against Acinetobacter baumannii
(Table 3). All three preparations such as EAE (C), EAE (BC), EAE (SC),
had less activity towards the pathogens such as Klebsiella pneumoniae
and Pseudomonas aeruginosa.
Considering the case of Chromobacterium violaceum WVAT6, all the
three preparations have better antibacterial activity against
Acinetobacter baumannii and moderate activity against Bacillus subtilis
and Staphylococcus aureus. Chromobacterium violaceum WVAT6 pre-
parations were not effective against the pathogens Salmonella typhi and
Salmonella paratyphi (Table 3).
3.4. Scanning electron microscopic (SEM) analysis of Chromobacterium
violaceum strain WVAT6 and Burkholderia sp. strain WYAT7
In SEM analysis, Chromobacterium violaceum WVAT6 appeared as
rod in shape (Fig. 3a). Burkholderia sp.WYAT7 appeared in coccobacilli
shape and was also observed as cells embedded in pellicle-like sub-
stance. This represents the formation of biofilm (Fig. 3b).
3.5. Screening for enzyme production
3.5.1. Protease production
In the skim milk agar plates, both bacteria produced a zone of
clearance indicating extracellular protease production (Fig. 4b).
Erythromycin 10mcg Penicillin-G Vancomycin 10mcg Tetracycline 10mcg
(E) 2units (P) (VA) (TE)
0(R) 0(R) 0(R) 12 mm(R)
13 mm(IM) 0(R) 0(R) 17 mm(IM)
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A. Ashitha, et al.
Fig. 6. Antibiotic sensitivity test. a - showing sensitivity of Burkholderia sp.WYAT7 to tetracycline and gentamicin, b-indicates sensitivity of Chromobacterium
violaceum WVAT6 to tetracycline, gentamicin, and erythromycin. 1-Penicillin-G, 2-Tetracycline, 3-Erythromycin, 4- Amoxicillin, 5- Vancomycin, 6- Gentamicin.
3.5.2. Screening for lipase production
The bacteria Burkholderia sp. WYAT7 and Chromobacterium viola-
ceum WVAT6 produced clear zone around the colonies in tributyrin
agar plates (Fig. 4c and d), which indicates lipase production.
3.6. Screening for biofilm production by Congo red agar method
The selected two endophytic isolates Chromobacterium violaceum
WVAT6 and Burkholderia sp. WYAT7 were identified as biofilm pro-
ducers as they formed black colored colonies in CRA medium (Fig. 5a).
Burkholderia sp. WYAT7 produced colonies with dry crystalline con-
sistency which indicated strong biofilm production (Fig. 5b). The plate
streaked with Aeromonas caviae (negative control) did not produced a
black colony on CRA plates (Fig. 5c).
3.7. Antibiotic sensitivity test of selected endophytes
The Chromobacterium violaceum WVAT6 was resistant to the anti-
biotics penicillin-G, amoxicillin, vancomycin where as sensitive to an-
tibiotics erythromycin, gentamicin and tetracycline (Table 4). In the
case of Burkholderia sp. WYAT7, it was sensitive against gentamicin and
tetracycline whereas resistant to other antibiotics tested (Fig. 6a).
4. Discussion
The Western Ghats harbors a great variety of plants which in-
corporate diverse species of endophytes [24,25]. Generally, the en-
dophytes isolated from the medicinal plants are mainly employed for
therapeutic purposes as they may contain or produce many of the
bioactive chemical compounds that are present in the host medicinal
plants. Artemisia nilagirica, a traditional medicinal plant has been re-
ported for its antimicrobial, antioxidant, anticancer, antiasthmatic and
larvicidal properties [17,26,27]. The plant is ubiquitously found in the
high altitude regions of Western Ghats especially in Wayanad region.
The endophytes are usually isolated from surface sterilized internal
tissues of plants derived from root, stem, leaves, etc [28]. Several stu-
dies have shown that a vast variety of endophytic bacteria can be iso-
lated from plant parts such as leaf, stem and root [29–31]. The extent of
use of sterilants and the method of sterilization have been reported to
influence the diversity of endophytes isolated [29,32].
The bacterial endophytes such as Pseudomonas spp., Serratia spp
.and Bacillus spp. dominated in the plant Artemisia nilagirica. Studies
have reported that Pseudomonas spp. and Bacillus spp. are the major
endophytic bacteria found associated with the majority of plants [33].
The plant Artemisia nilagirica also contain the endophytes such as
7
Microbial Pathogenesis 135 (2019) 103624
Chromobacterium violaceum, Burkholderia sp., Kosakonia cowanii, Ar-
throbacter sp.. Microbacterium sp., Psychrobacter sp and Enterobacter sp.
There are reports on Burkholderia sp. as the endophyte of different plant
species [34–36]. One of the studies reported that Coffea arabica L
contained Chromobacterium violaceum as an endophytic bacterium [37].
Quite a few studies have been reported on the endophytic association of
Chromobacterium violaceum in plants. This is the first study that reports
the bacterial endophytic association in the plant Artemisia nilagirica.
The antibacterial property plays a major role in therapeutic activ-
ities. In this study, the perpendicular streak method was used to de-
termine the antibacterial properties of the bacterial endophytes ob-
tained from Artemisia nilagirica against the selected human bacterial
pathogens. This method is considered as the preliminary qualitative
screening method for the antibacterial activity [19]. The study showed
maximum antagonistic activity against human pathogens by Chromo-
bacterium violaceum WVAT6 and Burkholderia sp.WYAT7.
The antagonistic activity reported in the present study might be due
to the presence of bioactive compounds present in the endophytes. So
methods were adopted for the extraction of antimicrobial compounds
from the endophytic bacteria. Ethyl acetate extract of the culture, ethyl
acetate extract of the boiled and cooled culture and the ethyl acetate
extract of the sonicated culture were tested for antibacterial activity.
The ethyl acetate extract of sonicated bacterial culture, EAE (SC), ex-
hibited a maximum zone of inhibition against most of the test patho-
gens. This may be due to the release of cell metabolites present inside
the bacterial cells [38]. The microbes synthesize the secondary meta-
bolites from a few precursors of primary metabolites with a relatively
small number. The endophytes generally produce low molecular weight
secondary metabolites such as antimicrobial compounds, vitamins like
B12 and B1, phytohormones or their derivatives and bioprotectants
[39–41]. Other secondary metabolites present in endophytes are alka-
loids, steroids, terpenoids, peptides, flavonoids, polyketones, phenols
and quinols [42]. In therapeutic applications, these compounds play a
significant roles as antimicrobial, antioxidant, anti-cancer, anti-in-
flammatory and immunosuppressive agents [43].
For the characterization of Chromobacterium violaceum WVAT6 and
Burkholderia sp.WYAT7, the bacteria were visualized under scanning
electron microscope to reveal the characteristic size and shape of the
cells. Chromobacterium violaceum WVAT6 showed typical rod shape,
while Burkholderia sp.WYAT7 was observed as coccobacillus in shape. It
has been reported that an environmental isolate of Burkholderia pseu-
domallei grown in soil has shown morphological alteration from rod
shape to coccoid [44] whereas a strain of Burkholderia paludis sp. ob-
tained from soil in Malaysia was observed as rod shaped [45].
Both Burkholderia sp.WYAT7 and Chromobacterium violaceum
A. Ashitha, et al.
WVAT6 were excellent protease producers. Study on Burkholderia
pseudomallei ATCC strain suggested that the expression of high pro-
teolytic activity may influence its pathogenic role [46]. Different strains
of Burkholderia both environmental and clinical isolates have been re-
ported to produce lipase enzyme [47–49]. Different Chromobacterium
strains have been reported with protease production [50,51]. The
present study revealed the lipase activity in both bacteria.
The peculiar characters such as biofilm formation may help the
bacteria in migration and effective colonization in the host plant.
Bacteria residing in the biofilm are protected by the exopolysaccharides
(EPS) [8]. During irreversible adhesion of bacterial cells to the host, the
protein profile of the bacteria would change and induce the production
of biofilm, further lead to the colonization of the bacteria. The bacterial
cell envelop and the protein secreted by them play a key role in bac-
terial cell physiology and interactions with their environment [52,53].
Biofilm also protects from environmental stresses like pH shift, osmotic
shock, UV radiation, toxins and different metal ions which are se-
questered in the presence of EPS [54]. An innate characteristic of bio-
film is antibacterial resistance and can also avoid host immune re-
sponses. In the present study, Burkholderia sp.WYAT7 and
Chromobacterium violaceum WVAT6 were found to produce biofilm ef-
fectively. Several studies have reported the colonization and biofilm
formation efficacy of bacteria on stem, leaves and rhizosphere of plants
[55,56]. The biofilm formation of endophytic bacteria can be stimu-
lated by plant root exudates, induction of stress conditions or anti-
microbial products. Steddom et al. [57], reported that bacteria can
colonize the mycelium of fungi and feed on its exudates, thus forms a
protective biofilm that prevents new growth of pathogens.
The bacteria generally acquire antibiotic resistance by natural
phenomenon for adaptation to adverse environments. In most of the
cases, the resistance to antibiotics by bacterial species is due to specific
target modification mechanism [58]. The biofilm production by the two
bacteria Burkholderia sp.WYAT7 and Chromobacterium violaceum
WVAT6 in the present study enhanced the multiple drug resistance of
the bacteria. These characters may augment the high altitude adapta-
tion of the bacteria, especially in the Wayanad region. Out of 13 isolates
of Chromobacterium violaceum isolated from oysters of Mexico, only one
was resistant to ampicillin and all were sensitive to gentamicin [59].
The antibiogram of clinical isolates of Chromobacterium violaceum was
reported as resistant to ampicillin, polymixin B, colistin sulfate [60].
Earlier strains of Burkholderia have been reported with multi drug re-
sistance by Zhou et al., [61]. Also, the study conducted by Hassan et al.
[62] reported that antibiotic resistance was higher in biofilm producing
bacteria than non-biofilm producers. Nair et al. [63] reported that the
pigmented bacteria have more antibiotic resistance than the non-pig-
ment producers. The isolates, Burkholderia sp.WYAT7 and Chromo-
bacterium violaceum WVAT6 examined in the current study were biofilm
and pigment producers with multidrug resistance and antimicrobial
activity.
5. Conclusion
All observations in this study showed that the medicinal plant
Artemisia nilagirica is a source of numerous bacterial endophytes, which
can produce several bioactive compounds with antibacterial properties.
The earlier works have reported the antibacterial property of the plant
Artemisia nilagirica. It has been reported that the bacterial endophytes
may produce the same or similar bioactive compounds as their host
plant. Studies are currently underway to determine the specific bio-
molecules responsible for the antibacterial property of the plant and its
endophytes. The antibacterial compounds obtained can be utilized for
the development of drugs in therapeutics. Also, their potential as a
source of agents against plant pathogens can be investigated as these
endophytes may have a natural role in the defense of plant against the
invading plant pathogens.
8
Microbial Pathogenesis 135 (2019) 103624
Declaration of conflict of interest
The authors declare no conflict of interest.
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