Aloha

Volume 3 Issue 1 {February 2023} ISSN: 2811 - 1451
SCREENING ACTINOMYCETES ASSOCIATED WITH LEMON GRASS

(CYMBOPOGON CITRATUS) RHIZOSPHERE FOR ACTIVITY
AGAINST MULTI DRUG RESISTANT BACTERIA
Ejiro Akponah
Department of Microbiology, Delta State University,
Abraka, Nigeria.
Monday Ubogu
Department of Microbiology, Federal University of Agriculture Makurdi,
Makurdi, Nigeria.
Email: ubomon@yahoo.co.uk.
ABSTRACT
Globally, there is increasing demand for novel bioactive molecules to combat the upsurge of
multiple antibiotics resistant pathogenic bacteria. Consequently, researches are now directed
towards different environments to identify microorganisms with capability of producing new,
potent and safe compounds. In this study, sixty Actinomycetes isolates obtained from the
rhizosphere of lemon grass were screened for antibacterial substance production using thirty-four
multiple antibiotic resistant bacteria isolates isolated from soils obtained from dumpsites in Abraka
metropolis, Delta State. Out of the 60 Actinomycete isolates, 6 (10%) belonging to the genus
Streptomyces demonstrated antibacterial activity against at least one of the 34 test bacterial
isolates. Although, 5(83.3%) out of the 6 Streptomyces spp exhibited antibacterial activity against
only the Gram positive test bacterium, 1(2.04%) coded as Str1, demonstrated broad spectrum
activity. This resulted in the extraction of its secondary metabolites using ethyl acetate. There were
no significant differences at p < 0.05 between zones of inhibition (ranging from 19 to 24 mm)
produced against various test isolate by the crude extract and that produced by the antibiotics used
as control (ceftriaxone) indicating susceptibility of the strains to the crude extract. Analysis of the
extract using GC-MS, led to the identification of 25 compounds. However, the main constituent
with suspected antibacterial activity were 1,2 benzodiol,3,5-bis(1,1-dimethylethyl)-, 2-methyl-7-
phenyl indole, 2,4,6, cycloheptatrien-1-one3,5, -bis-tri-methylsilyl and benzo[h]quinolone,2,4-
dimethyl. Hence, the study concludes that the Streptomyces sp Str1 produced compounds with
antibacterial properties against both Gram positive and negative multiple antibiotics resistant
bacteria and thus is a potential candidate for the development of promising antibacterial agents.
KEYWORDS: Actinomycetes, Bacteria, Lemon grass, Multidrug resistant, Rhizosphere,
Screening.
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RSU Journal of Biology and Applied Sciences (RSUJBAS)
INTRODUCTION
Over the last few decades, there has been increasing reports of bacterial resistance to various
antibiotics globally (Danquah et al., 2022; Al-Ansari et al., 2019; CDC, 2019; Manimaran et al.,
2017; Akponah, 2014). The major drivers of such antibiotic resistance have been attributed to both
overuse and misuse of antibiotics (Suganya et al., 2022; WHO, 2021a). The quantum of antibiotics
indiscriminately used for prophylactic and therapeutic measures in both man and farm animals
have resulted in the selection of resistance to multiple antibiotics in several pathogenic bacteria
(Al-Ansari et al., 2019; Nikaido, 2009). Pertinently, horizontal transmission of antibiotic resistant
genes has also been reported among environmental bacterial isolates and in agricultural animal
products (Suganya et al., 2022; Vinayamohan et al., 2022).
Multiple antibiotics resistance (MDR) is a global public health threat that need to be tackled in
order to achieve the sustainable Developmental goals of the United Nations (Suganya et al., 2022;
WHO, 2021b). In furtherance to this, resistance to antibiotics and other antimicrobials, play a key
role in the economy of any nation. It births the need for more expensive drugs hence draining the
finances of those impacted as well as the government, especially, in countries where government
attention is paid to subsidies in the health sector (WHO, 2021b). Antimicrobial resistance has also
resulted in severe/or difficult to threat infections, prolonged stays in hospitals, disabilities and even
death of teeming population irrespective of age (Rammali et al., 2022). This is very significant
because, the productivity of patients including their attendants are greatly impeded. Also, many
medical procedures like surgeries, organ transplantation, cancer chemotherapy have become very
risky as a result of the resistance of bacteria to various antibiotics (Hummell and Kirienko, 2020;
Rammali et al., 2022). In fact, the cost of multiple antibiotics resistance cannot be over-
emphasized. Therefore, there is an urgent need for the development of new antibiotics to forestall
the problem exerted by multi drug resistance in our modern day society (Danqua et al., 2022;
Mapipa et al., 2021; WHO, 2021b; Hummel and Kirienko, 2020; CDC, 2019).
In 2021, WHO reported a prevalence of 12.11%, 64%, 92.9%, 79.4% in methicillin resistant
Staphylococcus aureus, fluoroquinolone resistant Escherichia coli and carbapenem resistant
Klebsiella pneumonia in countries that report to the Global Antimicrobial Resistance and Use
Surveillance System (GLASS). Recently, the US Center for Disease and Prevention issued a health
advisory to warn the public of an increase in drug resistance in bacteria since there are limited
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recommendations for treatment for infections caused by such bacteria. According to the CDC
(2019) report, percentage of infections caused by drug resistant strains of Shigella increased from
0 in 2015 to 5% in 2019. Furthermore, the report states that there are nearly 3 million antimicrobial
resistant infections each year and more than 35000 people die as a result in the US and about 5
million deaths recorded worldwide. These records are expected to increase to 10 million by 2050
if efforts are not made to stop the spread of antimicrobial resistance (Santos-Beneit et al., 2022).
The rapid onset of resistance to several drugs, requires a constant supply of new drugs for the
effective treatment of infections (Arya et al., 2019; Al-Ansari et al., 2019; Manimaran et al., 2017;
Gill et al., 2011). Thus, screening and discovery of novel drugs have become a necessity (Danquah
et al., 2022; Manimaran et al., 2017; Parsaeimehr et al., 2013). Natural products play a pivotal
role in antibiotic drug discovery (Rammali et al., 2022) and microorganisms are considered as
reservoir of such natural products and hence untapped antimicrobials, (Danquah et al., 2022;
Shukla, 2015). They offer a plethora of new, cheap, alternative bioactive compounds that can be
used excellently to combat the upsurge of pathogenic antibiotic resistant strains (Danquah et al.,
2022 Rakholiya et al., 2013).
Microbial metabolites produced especially by Actinomycetes have been the basis for the synthesis
of many antibiotics (Promnuan et al., 2020) hence making this group of organisms highly
important in the medical and pharmaceutical sectors. The metabolites of Actinomycetes have not
only exhibited antimicrobial activity but have also shown anti-tumor, anti-viral, anti-cancer anti-
inflammatory as well as immunosuppressive activities (Rammali et al., 2022; Gomes et al.,2018;
Shukla, 2015). Actinomycetes are a group of Gram positive, acid fast, facultatively anaerobic,
filamentous bacteria possessing the tendency to break or fragment into cocci or bacillary forms
(Gottelt et al., 2010). The morphological complexity and diversity of this group put them in
limelight as they are widely distributed in both aquatic and terrestrial environments (Selim et al.,
2021) and are well known sources of an array of bioactive secondary metabolites (Promnuan et
al., 2020; Al-Ansari et al., 2019; Ramanzani et al., 2013; Gottelt et al., 2010). More than 50 % of
the explored bioactive molecules have been produced by Actinomycetes (Gomes et al., 2018;
Shukla, 2015) hence, the group stay indispensable in the race to ameliorate the impact of drug
resistant pathogens (Mast and Stegmann, 2019).
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As crucial as the foregoing, there has been a decrease in the discovery of new bioactive compounds
from actinomycetes in the last two decades (Santos-Berneit et al., 2022; Mojica et al., 2021; WHO,
2021b). Therefore, researches are now, re-directed towards unexploited environments (Santos-
Berneit et al., 2022; Manimaran et al., 2017) to obtain Actinomycetes with potentials for vast and
new compounds that would be able to effectively and safely target resistant pathogens (Rammali
et al., 2022) To this end, this study was designed to screen lemon grass rhizosphere ( a culinary
and medicinal grass common in the Southern part of Nigeria) for common soil-derived/indigenous
Actinomycetes capable of producing bioactive compound(s) with activity against multi-drug
resistant bacteria.
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MATERIALS AND METHOD
Source of Samples/Actinomycetes isolates
Rhizosphere samples were obtained from 30 lemon grasses growing at three different sites
(designated Station A, B and C) in Abraka, Delta State following the method described by Ubogu
et al., 2019 and Kumar et al., 2012. Loose soils around the root of each uprooted grass, was shaken
off while soil firmly attached to the root was carefully but vigorously shaken into sterile black
polyethylene bag. After which, the rhizosphere soil samples were transported to the laboratory
within 30 minutes of collection for isolation of Actinomycetes.
The Actinomycete isolates were obtained by culture method. Each rhizosphere sample was
weighed in 1 g amount and diluted using the ten-fold serial dilution method. Thereafter, 0.1 mL of
various dilution factors were inoculated separately into freshly prepared starch casein agar using
the pour plate method. Incubation at ambient temperature for 48-72 h, followed immediately. At
the end of incubation period, plates of an appropriate dilution factor that produced distinct colonies
ranging from 30 -300 were selected for isolation and further studies. The composition of the starch
casein agar in g/L is as follows: FeSO4.7H2O; 0.01, MgSO4.7H2O; 0.05, CaCO3; 0.02, NaCl; 2.00,
KNO3; 2.0, K2HPO4; 2.0, casein; 0.3, starch 10, and agar 15.
Identification of Actinomycete Isolates
Preliminary identification of isolates was based on observation of cellular and colonial

morphologies. Isolates were further characterized biochemically by subjection to criteria as in
Bergey’s Manual of Determinative Bacteriology (Goodfellow et al., 2012).
Source of ‘Test Bacteria’ Isolates
Multiple antibiotics resistant bacteria (MDRB) isolates obtained from dumpsite soils (4) receiving
several municipal wastes served as the test organisms. After careful removal of all solid waste,
composite soil sample at a depth of 5cm (vertical profile) was collected from the dumpsites using
sterile soil auger and transported in black polyethylene bags to the laboratory within 30 minutes
of collection. Bacterial isolates were obtained by diluting 1g of each soil sample using the ten-fold
serial dilution method and then inoculating 0.1mL aliquot of appropriate dilution factor into Muller
Hinton agar. Plates were then incubated at room temperature for 24 to 72 h. The isolates obtained
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were characterized biochemically according to criteria in Bergey’s manual of determinative

bacteriology (1994) and multidrug resistance determined by antibiotic susceptibility test as
described below.
Screening test bacteria for antibiotic Susceptibility.
The Kirby-Bauer disc diffusion method according to the Clinical Laboratory Standards Institute
(CLSI) guidelines was adopted. Each test bacterium was standardized by sub-culturing 3 colonies
of the respective isolate into sterile peptone water contained in a test tube and incubated at 37OC
until turbidity marched that of 0.5 BaSO4 (McFarland standard). The standardized inoculum was
then inoculated using a sterile swab stick onto the surface of freshly prepared Muller Hinton agar.
Then, plates were allowed to stand for about 5 minutes and various antibiotic discs were aseptically
placed on the surface of the already seeded agar plate. The antibiotics used to assess the sensitivity
of the isolates were septrin (30µg), chloramphenicol (30 µg), sparfloxacin (10µg), ciprofloxacin
(10µg), augmentin (30µg), amoxicillin(30µg), gentamycin (10 µg), streptomycin (30 µg),
cefuroxime (30 µg) and ceftriaxone (30 µg). Incubation followed immediately at 37OC for 18 -24
h. At the end of incubation, zones of inhibition were measured in mm and interpreted according to
CLSI (2018) guideline. Isolates that were resistant to three or more classes of antibiotic were
considered as MDR and were selected as the test bacteria. Also, percent occurrence (prevalence)
of MDRB were determined.
Preliminary Screening of Streptomyces spp. for the production of Antibacterial Compounds
Preliminary screening for the production of bioactive substance (antibacterial) was done using the
cross streaking method. Each of sixty Actinomycete isolates as obtained from lemon grass
rhizosphere, was streaked across one-third portion of freshly prepared nutrient agar plates and
incubation at room temperature for 7 days followed immediately. After which, the test bacterium
(MDRB) was streaked perpendicularly, to the already grown Actinomycete isolate. Further
incubation followed immediately at 37oC for 24 h. At the end of incubation period, zone of
inhibition was measured and regarded as indication for the production of bioactive substance with
antibacterial activity.
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Secondary Screening of Streptomyces spp. for the production of Antibacterial Compounds
The Actinomycete strain that elicited significant zone of inhibition against at least one strain of
each MDR bacterium was selected for the secondary screening experiment. The experiment was
done following a two-step procedure. In the first step, crude extract of the metabolites of the
Actinomycete was obtained while the second step comprised evaluation of the antibacterial
activity of the crude extract as described in the succeeding sections.
Production of crude extract of the metabolites from Streptomyces spp.
The choice strain of Actinomycete (Streptomyces sp.) was subjected to fermentation in starch
casein broth. Cell concentration of 1.00 x 107 CFU/mL was inoculated into 100mL of the broth
contained in a 250mL Erlenmeyer flask. Then, incubation at 30oC for 7 days under shaken
conditions of 120 rpm using shaker incubator (SEARCHTECH) was done immediately. Crude
extracts of the metabolites produced was then, obtained from the supernatant using 1% v/v ethyl
acetate following the method described by Dhananjeyan et al., 2010. Having harvested the cells,
ethyl acetate was mixed with the supernatant in ratio 1:1. The mixture was allowed to stand
overnight, after which the separated upper layer of ethyl acetate was evaporated to dryness at 40oC.
Concentrated bioactive compounds was reconstituted in sterile deionized water for further
analysis.
Determination of the anti-bacterial activity of crude extract of metabolites produced by

Streptomyces sp.
The paper disc diffusion technique was used in the determination of the anti-bacterial activity of
the crude extract obtained as described in the preceding section. The Kirby –Bauer disc diffusion
method was adopted. Paper disc (3mm in diameter were cut from Whatman No 1 filter paper. After
which they were allowed to cool and impregnated with 0.1mL of 0.01mg/L of reconstituted crude
extract of bioactive compounds. This was then aseptically placed on the surface of an already
seeded Muller Hinton agar medium. Seeding was done by respective streaking of standardized
MDR bacterial isolate all over the surface of each medium. (The MDR bacteria used in this
experiment included only the isolates that demonstrated susceptibility to anti-bacterial substance
producing Streptomyces in preliminary screening) The antibiotics ceftriaxone was used as control.
7
All plates were incubated immediately at 37oC for 24h. At the end of incubation period, zones of
inhibition produced were measured.
Determination of the bioactive compounds present in crude extract.
Investigation to identify the bioactive compounds present in the ethyl-acetate extract obtained as
described above, was done using gas chromatography coupled with mass spectrometer detector
(Agilent Technologies). Compound identification was achieved based on reference to mass spectra
database in NIST (National Institute of Standard and Technology) library.
Statistical Analysis
Data obtained were expressed as mean and percentage. Statistical significance was assessed by t-
test and p < 0.05 was considered as statistical significance.
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RESULTS
Two hundred and two bacteria isolates belonging to five genera including Salmonella,
Escherichia, Pseudomonas, Acinetobacter and Staphylococcus were obtained from soil samples
collected from dump sites. Upon screening for antibiotic susceptibility, varied degrees of
sensitivity to the antibiotics used in the assay were noticed in the 210 isolates. it was observed that
34 (16.8%) of the 202 bacterial isolates demonstrated multiple antibiotic resistance as presented
in Table 1. The prevalence of multiple antibiotic resistance observed among isolates of
Staphylococcus aureus (16), Acinetobacter spp. (36), Salmonella spp. (51), Escherichia coli (69),
and Pseudomonas aeruginosa (30) were 37.5, 13.9, 9.8, 26.7 and 14.5 (%) respectively (Table 1).
On the basis of the multiple antibiotic resistance, these 34 bacterial isolates were selected as test
organisms for preliminary screening of various Actinomycetes isolates for potential of anti-
bacterial substance production.
Furthermore, a perusal of the data as presented in Table 2, revealed that at least 15% of the isolates
of the various test bacteria (Pseudomonas aeruginosa, Acinetobacter spp., Salmonella spp.,
Escherichia coli and Staphylococcus aureus) were resistant to sceptrin, chloramphenicol,
sparfloxacin, ciprofloxacin, gentamycin, streptomycin and amoxicillin. However, all isolates with
the exception of Pseudomonas aeruginosa, were susceptible to ceftriaxone (hence its choice as
control antibiotics in subsequent secondary screening experiment). Two strains (6.7%) of
Pseudomonas aeruginosa demonstrated resistance to ceftriaxone. Also, most isolates were
susceptible to augmentin though percentages of population observed as resistant to it ranged from
0 - 8.3.
A total of sixty Actinomycetes isolates were obtained from lemon grass rhizosphere soils, of
which, 49 (81.67%) were Streptomyces spp. Micromonospora and Nocardia were 5 (8.33%) and
6 (10%) respectively as presented in Table 3. Out of these 60 Actinomycete isolates, 6 (10%)
belonging only to the genus Streptomyces demonstrated antibacterial activity against at least one
of the 34 MDR bacterial isolates as depicted in Table 4. The results of the preliminary screening
revealed that, 5(83.3%) out of the 6 Streptomyces spp. that exhibited antibacterial activity, were
more active against the Gram-positive test bacterium than the Gram-negative bacteria (Table 5).
However, 1(2.04%) demonstrated activity against all the MDRB used in the assay as also,
presented in Table 5.
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In addition, the Streptomyces strain coded as Str1 was the only Streptomyces species capable of
producing significant zones of inhibition against at least one isolate of each of the five genera of
MDR test organisms irrespective of Gram response. Consequently, its metabolites were extracted
following fermentation in starch casein broth. The results of the anti-bacterial activity of the extract
from Str1, is as presented in Fig. 1. The crude extract produced zones of inhibitions ranging from
23-25(mm), 19-21(mm), 21-23(mm), 20-24(mm) and 22-24(mm) against Staphylococcus aureus,
Acinetobacter spp., Pseudomonas aeruginosa, Escherichia coli and Salmonella spp. respectively.
Again, broad spectrum activity was noticed and zones of inhibition were all greater than or equal
to zones of inhibition produced by the control antibiotics. There were no significant differences at
p < 0.05 between zones of inhibition produced against each isolate by the crude extract and that
produced by the antibiotics used as control indicating susceptibility of the strains to the crude
extract. The ethyl acetate extract demonstrated better inhibitory properties against the multidrug
resistant bacteria than all the assayed antibiotics except ceftriaxone and augmentin.
Analysis of the extract using GC-MS, led to the identification of 25 compounds (25 peaks).
However, the main constituent with suspected antibacterial activity as shown in Table 6, were 1,2
benzodiol,3,5-bis(1,1-dimethylethyl)-, 2-methyl-7-phenyl indole, 2,4,6, cycloheptatrien-1-one3,5,
-bis-tri-methylsilyl and benzo[h]quinolone,2,4-dimethyl. The resemblance of the mass spectrum
of each of these compounds to the structure in the reference library were greater than 70% while
their area ranged from 6.202 to 8.630 (%). All of these biomolecules are heterocyclic containing
the benzenoid nucleus and their elution periods lasted between 12.984 and 16.687 minutes.
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Table 1: Prevalence of multiple antibiotic resistant bacteria among isolates screened for antibiotic
susceptibility
Organism Number isolates Number of Isolates Prevalence of

from dump sites that were MDR MDR Isolates (%)
S. aureus 16 6 37.5
Acinetobacter sp. 36 5 13.9
Salmonella sp. 51 5 9.8
P. aeruginosa 30 8 26.7
E. coli 69 10 14.5
Total 202 34 16.8
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Table 2: Antibiogram of isolates obtained from dumpsite Soils
Antibiotic Number of Resistant isolates (percentage)

P. aeruginosa Acinetobacter Salmonella E. coli S. aureus
spp. spp.
SEP 15(50.0%) 11(30.6%) 15(29.4%) 16(23.2%) 8(50.0%)
CHL 11(36.7%) 9(25%) 8(15.7%) 13(18.8%) 3(18.8%)
SPA 10(33.3%) 12(33.3%) 10(29.4%) 14(20.3%) 6(37.5%)
CIP 11(36.7%) 12(33.3%) 13(19.6%) 14(20.3%) 6(37.5%)
AUG 2(6.7%) 3(8.3%) 0(0%) 4(5.8%) 1(6.3%)
AMX 9(30.0%) 14(38.9%) 18(35.3%) 6(8.7%) 7(43.8%)
GEN 7(23.3%) 6(16.7%) 14(27.5%) 13(18.8%) 2(12.5%)
STR 8(26.7%) 6(16.7%) 15(29.4%) 11(15.9%) 2(12.5%)
CEF 2(6.7%) 2(5.6%) 0(0%) 0(0%) 1(6.3%)
CER 2(6.7%) 0(0%) 0(0%) 0(0%) 0(0%)
SEP=Sceptrin, CHL=Chloramphenicol, SPA=Sparfloxaci, CIP= Ciprofloxacin, AUG=
Augmentin, GEN= Gentamycin, STR= Streptomycin, CEF = Cefuroxime, CER= Ceftriaxone,
AMX = Amoxicillin
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Table 3: Frequency of occurrence of various Actinomycetes in the rhizosphere of Lemon

grass from different stations
Actinomycetes Number of isolates obtained from lemon grass Total

rhizosphere.
Station A Station B Station C
Streptomyces spp. 14 16 19 49(81.7%)
Micromonospora spp. 3 2 0 5(8.3%)
Nocardia spp. 3 2 1 6(10%)
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Table 4: Percentage of Actinomycetes that demonstrated antibacterial activity.
Actinomycetes (n) Percentage that Possessed antibacterial activity

Streptomyces spp. (49) 12.24
Nocardia spp. (6) 0
Micromonospora spp. (5) 0
Total (60) 10
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Table 5: Response of various strains of MDRB to inhibitory action of antibacterial substance

producing Streptomyces spp.
Antibacterial Proportion of Susceptible MDRB (%)

Producing
Streptomyces
Isolates
Staphylococcus Acinetobacter Pseudomonas Escherichia Salmonella
aureus (n= 6) spp. (n = 5) aeruginosa (n coli (n = 10) spp. (n = 5)
= 8)
Str1 50 100 50 60 60
Str2 16.7 0 12.5 10 0
Str3 16.7 0 12.5 0 0
Str4 50 0 0 0 0
Str5 33.3 0 0 10 0
Str6 33.3 0 12.5 10 0
15
Strain1 strain 2 strain 3

strain 4 strain 5 strain 6
control
30
25
Mean Zone Inhibition (mm)
20
15
10
0
Staphylococcus Acinectobacter Pseudomonas Escherichia coli Salmonella spp
aureus spp aeruginosa
MDRB Isolates
Fig.1: Antibiogram of Crude Extract obtained

from Streptomyces sp (Str1)
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Table 6: Chemical composition of compounds in ethyl acetate extract with antibacterial activity.
S/N Compound Retention Peak % Correlation with Area

time Height compound in %
(mins) library
1 1,2 benzodiol,3,5- 12.984 2783 71.84 6.202

bis(1,1-dimethylethyl)-
2 benzo[h]quinolone,2,4- 14.555 2931 79.89 6.895

dimethyl
3 2,4,6,cycloheptatrien- 15.504 4109 95.53 8.245

1-one3,5,-bis-tri-
methylsilyl
4 2-methyl-7-phenyl 16.687 4970 100 8.630

indole
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Fig.2: GC-MS chromatogram of the ethyl-acetate extract.
18
DISCUSSION
Multiple antibiotic resistance has been defined as the acquisition of resistance to at least any three
groups or classes of antibiotics (Suganya et al., 2022; Bjerketorp et al., 2021; Exner et al., 2017).
According to Tanwar et al., 2014, multiple antibiotic resistance is defined as insensitivity of
microorganisms to administered antibiotics (which are structurally unrelated and have different
molecular targets) despite previous sensitivity to the drugs. Therefore, the isolation of MDRB from
dumpsite soil samples is alarming and may be connected to the habitual indiscriminate disposal of
all kinds of materials including hospital waste, animal farm waste and the likes that probably
contain unused, expired or residual antibiotics. Also, the incessant use of sub- therapeutic doses of
antibiotics by the people and in livestock production as well as the problem of rampant open
defecation may have contributed to the pollution of the environment by antibiotics and hence the
occurrence of MDRB in the environment studied. Similar reports have been made by Mokni-Tlili
et al., 2023 and Nikaido, 2009. In addition, the microbial production of secondary metabolites that
resemble many antibiotics currently in use in health care systems could also have promoted the
observed incidence of MDRB among the environmental isolates as a direct consequence of
selective pressure. Again, the dissemination of antibiotic resistance genes among the isolates in
the environment can be attributed to horizontal transfer of antibiotic resistant genes among
bacterial within same ecological niche (Vinayamohan et al., 2022). Several authors have shown
the presence of plasmids and integrons (mobile genetic elements) that carry antibiotic resistance
gene among and across species of bacteria in the environment (Suganya et al., 2022; Vinayamohan
et al., 2022).
Further results obtained in this study, confirms the abundance of Streptomyces spp in the
rhizosphere of lemon grass as it was the predominant species of Actinomycetes in samples
collected from different stations. Several authors including Chaudhary et al., 2013, Ramazani et
al., 2013 and Alimuddin et al., 2011 have reported the predominance of Streptomyces among other
Actinomycetes in soils and hence rhizospheres. This is usually linked to soils with near neutral
and alkaline pH, high humic and moderate moisture contents (Sapkota et al., 2020; Ramazani et
al., 2013; Nanjwade et al., 2010; Hayakawa, 2008). The type of culture medium used in isolation
can also favour a particular species of Actinomycetes (Rammali et al., 2022; Promnuan et al.,
2020). In this case starch casein agar was used.
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Among the Streptomyces, the rate of isolation of species with antibacterial property was 12.24%,
although, only 2.04% demonstrated activity against all the MDRB used in the assay (ie broad
spectrum activity). Both higher and lower percentages have been reported in previous studies
(Promnuan et al., 2020; Manimaran et al., 2017; Ramazani et al., 2013). Moreover, the lower
susceptibility of Gram-negative test bacteria than the Gram-positive bacterium to the antibacterial
activity of majority (83.3%) of Streptomyces spp. assayed in the preliminary screening probably,
relates to the nature of their cell walls. The Gram negative bacterial cell wall contain phospholipids
and lipopolysaccharides which make it impermeable to hydrophilic substances (Rammali et al.,
2022). The extent of the antimicrobial capabilities of organisms is dependent on the kind of
secondary metabolite produced and relies ultimately, on its genetic machinery (Bjerketorp et al.,
2021; Shukla, 2015). This may explain the variability noted in the antagonistic activities of the
various Streptomyces species against the test bacteria.
Interestingly, the ethyl acetate extract of the metabolites of the promising Streptomyces strain,
demonstrated better inhibitory property to the MDRB than sceptrin, chloramphenicol,
ciprofloxacin, sparfloxacin gentamycin and streptomycin. This is most likely due to the occurrence
of compounds in the extract with different chemical structure than these antibiotics. The extract
was shown to contain 2-methyl-7-phenyl indole, 2,4,6, cycloheptatrien-1-one3,5, -bis-tri-
methylsilyl 1,2 benzodiol,3,5-bis(1,1-dimethylethyl)- and benzo[h]quinolone,2,4-dimethyl. This
result is consistent with those of other investigators who have reported a wide array of secondary
metabolites with antimicrobial activities, produced by different species of Streptomyces (Ghanem
et al., 2022; Promnuan et al., 2020; Al-Ansari et al., 2019; Manimaran et al., 2017).
Literature survey has demonstrated that indole derivatives have diverse biological activities
including anti-bacterial, anti-viral, anti-plasmodium, antioxidant, anti-inflammatory and anti-
cancer (Seenivasan et al., 2022; Vicham et al., 2022; Kumar and Ritika, 2020; Arora et al., 2018;
Ambrus et al., 2009). Therefore, the production of 2-methyl-7-phenyl indole as one of its
secondary metabolites, may have contributed to the anti-bacterial tendency displayed by the
Streptomyces sp. Str1. Also, some authors have demonstrated the effectiveness of synthesized
2,4,6, cycloheptatrien-1-one3,5,-bis-tri-methylsilyl as well as benzoquinoline and its derivatives
against both Gram positive and Gram negative bacteria (Sani, 2022; Antoc et al., 2021; Haiba et
al., 2016). Probably, these compounds which are more or less aromatic are likely responsible for
20
the broad-spectrum activity of the strain of Streptomyces assayed and its extract. Although, these
compounds have been chemically synthesized and their antibacterial properties elucidated, they
have not been previously reported in Streptomyces from lemon grass rhizosphere.
Finally, the study concludes that the Streptomyces sp. Str1 produced compounds with antibacterial
properties against MDRB and thus is a potential candidate for the development of promising
antibacterial agents. However, there is need for further research on the purification of these
compounds for the determination of their mechanisms of action, in vivo activities including their
pharmacokinetics, pharmacodynamics and cytotoxicity prior to their inclusion in the repertoire of
bioactive substances produced by Streptomyces species.
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Volume 3 Issue 1 {February 2023} ISSN: 2811 - 1451

SCREENING ACTINOMYCETES ASSOCIATED WITH LEMON GRASS

MATERIALS AND METHOD

Source of Samples/Actinomycetes isolates

Identification of Actinomycete Isolates

Preliminary identification of isolates was based on observation of cellular and colonial

Source of ‘Test Bacteria’ Isolates

were characterized biochemically according to criteria in Bergey’s manual of determinative

Screening test bacteria for antibiotic Susceptibility.

Preliminary Screening of Streptomyces spp. for the production of Antibacterial Compounds

Secondary Screening of Streptomyces spp. for the production of Antibacterial Compounds

Production of crude extract of the metabolites from Streptomyces spp.

Determination of the anti-bacterial activity of crude extract of metabolites produced by

Determination of the bioactive compounds present in crude extract.

Organism Number isolates Number of Isolates Prevalence of

Table 2: Antibiogram of isolates obtained from dumpsite Soils

Antibiotic Number of Resistant isolates (percentage)

Table 3: Frequency of occurrence of various Actinomycetes in the rhizosphere of Lemon

Actinomycetes Number of isolates obtained from lemon grass Total

Table 4: Percentage of Actinomycetes that demonstrated anti- bacterial activity.

Actinomycetes (n) Percentage that Possessed antibacterial activity

Table 5: Response of various strains of MDRB to inhibitory action of antibacterial substance

Antibacterial Proportion of Susceptible MDRB (%)

Strain1 strain 2 strain 3

Fig.1: Antibiogram of Crude Extract obtained

S/N Compound Retention Peak % Correlation with Area

1 1,2 benzodiol,3,5- 12.984 2783 71.84 6.202

2 benzo[h]quinolone,2,4- 14.555 2931 79.89 6.895

3 2,4,6,cycloheptatrien- 15.504 4109 95.53 8.245

4 2-methyl-7-phenyl 16.687 4970 100 8.630

Fig.2: GC-MS chromatogram of the ethyl-acetate extract.

Akponah, E. (2014). Occurrence of Plasmid Borne Multiple Antibiotic Resistant Genes in

World Health Organization (WHO) (2021a). Antimicrobial Resistance. www.who.int.

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