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Comparative Study on the Antibacterial Activities of Neem Oil, Mustard oil and Black Seed Oil Against Pathogenic Staphylococcus aureus, Klebsiella pneumoniae, Salmonella Typhi and Pseudomonas Aeruginosa

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Comparative Study on the Antibacterial Activities of Neem Oil, Mustard oil and Black Seed Oil Against Pathogenic Staphylococcus aureus, Klebsiella pneumoniae, Salmonella Typhi and Pseudomonas Aeruginosa

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The study was aimed to determine the antimicrobial activity of commercially available Neem oil, Mustard oil and Black seed oil against some disease causing organism such as Staphylococcus aureus, Klebsiella pneumoniae, Salmonella typhi and Pseudomonas aeruginosa. The antibacterial activity of these oil against selected pathogens were determined by dilution method, disc diffusion and agar well diffusion method. Pathogens were collected from a tertiary hospital and the pathogenicity was determined by DNAse, Coagulase and Blood Agar hemolysis test. All three essential oil tested showed antibacterial activity against Staphylococcus aureus, Klebsiella pneumonia and Salmonella typhi. Black seed oil and Neem oil showed the highest rate of antibacterial activity even at very low concentration. In case of all three pathogens, inhibition of growth caused by Neem oil was more than 99%. Black seed oil inhibited the growth of Staphylococcus aureus by 100% and by 99.97% in case of Klebsiella pneumonia. Mustard oil exhibited antibacterial activity against the tested bacteria by dilution method but no zone of inhibition was found by agar disc diffusion or agar well diffusion method. Pseudomonas aeruginosa exhibited very low degree of sensitivity to Mustard oil and Black seed oil. However, Neem oil showed antibacterial activity against Pseudomonas aeruginosa by dilution method only. The results showed that all these oils can be a good source of antibacterial agent. The encouraging results also indicate that these oils should be exploited as natural antibiotic for the treatment of many infectious diseases caused by these pathogens, and will be helpful in understanding the relations between ancient cures and current medicines.
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European Journal of Scientific Research
ISSN 1450-216X / 1450-202X Vol. 154 No 1 September, 2019, pp. 58-67
http://www. europeanjournalofscientificresearch.com
Comparative Study on the Antibacterial Activities of Neem Oil,
Mustard oil and Black Seed Oil Against Pathogenic
Staphylococcus aureus, Klebsiella pneumoniae, Salmonella Typhi
and Pseudomonas Aeruginosa
Sharmin Sultana
Student, Biotechnology Program
Department of Mathematics and Natural Sciences
BRAC University, Bangladesh
Nishat Anzum Shova
Student, Microbiology Program
Department of Mathematics and Natural Sciences
BRAC University, Bangladesh
Akash Ahmed
Lecturer, Microbiology Program
Department of Mathematics and Natural Sciences
BRAC University, Bangladesh
M. Mahboob Hossain
Professor, Microbiology Program
Department of Mathematics and Natural Sciences
BRAC University, Bangladesh
E-mail: mmhossain@bracu.ac.bd
Tel: 88-02-9844051 Ext. 4113; +8801715107660
Abstract
The study was aimed to determine the antimicrobial activity of commercially
available Neem oil, Mustard oil and Black seed oil against some disease causing organism
such as Staphylococcus aureus, Klebsiella pneumoniae, Salmonella typhi and
Pseudomonas aeruginosa. The antibacterial activity of these oil against selected pathogens
were determined by dilution method, disc diffusion and agar well diffusion method.
Pathogens were collected from a tertiary hospital and the pathogenicity was determined by
DNAse, Coagulase and Blood Agar hemolysis test. All three essential oil tested showed
antibacterial activity against Staphylococcus aureus, Klebsiella pneumonia and Salmonella
typhi. Black seed oil and Neem oil showed the highest rate of antibacterial activity even at
very low concentration. In case of all three pathogens, inhibition of growth caused by
Neem oil was more than 99%. Black seed oil inhibited the growth of Staphylococcus
aureus by 100% and by 99.97% in case of Klebsiella pneumonia. Mustard oil exhibited
antibacterial activity against the tested bacteria by dilution method but no zone of inhibition
was found by agar disc diffusion or agar well diffusion method. Pseudomonas aeruginosa
exhibited very low degree of sensitivity to Mustard oil and Black seed oil. However, Neem
oil showed antibacterial activity against Pseudomonas aeruginosa by dilution method only.
Comparative Study on the Antibacterial Activities of Neem Oil, Mustard oil and
Black Seed Oil Against Pathogenic Staphylococcus aureus, Klebsiella pneumoniae,
Salmonella Typhi and Pseudomonas Aeruginosa 59
59
The results showed that all these oils can be a good source of antibacterial agent. The
encouraging results also indicate that these oils should be exploited as natural antibiotic for
the treatment of many infectious diseases caused by these pathogens, and will be helpful in
understanding the relations between ancient cures and current medicines.
Keywords:
Natural Antimicrobial agent, Disc diffusion method, well diffusion
method, pathogenic bacteria
, zone of inhibition
1. Introduction
In this era of emergence of multi drug resistant organisms and a decrease in newer antibiotics, the
ancient healing methods by using traditional oil can be considered a very effective way. People's
perception towards traditional medicine has also changed and is very encouraging. Medicinal plants
have been used for curing diseases for many centuries in different indigenous systems of medicine as
well as folk medicines. Moreover, Ahmad et al.,(2013) showed medicinal plants are also used in the
preparation of herbal medicines as they are considered to be safe as compared to modern allopathic
medicines. World health organization estimates that 80% of the population living in the developing
countries relies exclusively on traditional medicine for their primary health care. Kumar and
Navaratnam (2013) investigated more than half of the world's population still relies entirely on plants
for medicines, and plants supply the active ingredients of most traditional medical products. Further,
Hulin et al.,(1998) stated higher and aromatics plants have traditionally been used in folk medicine as
well as to extend the shelf life of foods, showing inhibition against bacteria, fungi and yeasts. Most of
their properties are due to essential oils produced by their secondary metabolism. Essential oils and
extracts from several plant species are able to control microorganisms related to skin, dental caries, and
food spoilage, including Gram-negative and Gram-positive bacteria. Sartoratto et al.,(2004) described
many countries have maintained research programs to screen traditional medicines for antimicrobial
activity, as is the case of India, Palestine, Africa, Honduras, Jordan, Cuba and Italy . Plants from
Brazilian biomes have also been used as natural medicines by local populations in the treatment of
several tropical diseases, including schistosomiasis, leishmaniasis, malaria and fungal and bacterial
infections.
Nigella sativa of the Ranunculaceae family is a medicinal plant of traditional Indian medicine.
Al-Ali et al. (2008), found it possesses many pharmacological activities, also considered as one of the
peak forms of healing medicine, treatment of various diseases like bronchitis, diarrhea, rheumatism,
asthma and skin disorders, used in digestive disorders, to increase milk production in nursing mothers
to fight parasitic infection. Azadirachta indica is locally known as Neem. Akerele, (1993) has claimed
it is a tree in the mahogany family and native to India, Bangladesh, Thailand, Nepal and Pakistan.
Again, Ping et al.,(2002) has revealed neem is widely used for the treatment of incurable diabetes and
Prieto et al.,(2002) showed it can be used to control diseases such as leprosy, intestinal helminthiasis
and respiratory system. According to Britto et al.,(2013) it also have antiviral, antibacterial, antifungal,
anti-inflammatory, antipyretic, antiseptic and antiparalitic uses. Brassica juncea is locally known as
mustard and Grieve (1931) suggested it can be used in inflammation, chiefly in pneumonia, bronchitis
and other diseases of the respiratory organs. There are various infection causing pathogens like
Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Klebsiella pneumoniae are used
in this study to measure the efficiency of these natural oils.
Aims
60 Sharmin Sultana, Nishat Anzum Shova, Akash Ahmed and M. Mahboob Hossain
Aim of this study was to find the antimicrobial activity of Neem oil, Mustard oil and Black seed
oil against some pathogenic bacteria such as; Staphylococcus aureus, Klebsiella pneumoniae,
Salmonella typhi and Pseudomonas aeruginosa.
2. Materials and Methods
Bacterial Strains
In this study, the used bacterial species were Staphylococcus aureus, Pseudomonas aeruginosa,
Klebsiella pneumoniae, and Salmonella typhi. All these organisms were collected from a tertiary
hospital.
Product Tested
Commercially available Neem oil, Mustard oil and Black seed oil which do not contain any
preservatives.
Conformation of the Pathogenic Bacteria
Each bacterial strain were subjected to morphological and biochemical confirmation tests to determine
the purity of the samples. All the biochemical tests were performed in specific media according to the
standard methods described in Microbiology Laboratory Manual by Cappuccino and Natalie (2013).
All the bacterial cultures were grown on nutrient agar plates in the incubator at 37C before the process
of any biochemical identification test. Lastly, the pathogenicity was determined by DNAse, Coagulase
and Blood Agar hemolysis test
Preparation of Stock Sample
For short-term preservation, 2 ml of T1N1 agar butt in a vial was inoculated by stabbing bacterial
growth of each isolate from nutrient agar plate. Then the vial was kept at 4 ºC for an hour to gelatinize.
After an hour, the surface of the medium was covered with sterile paraffin oil and the vial was stored at
room temperature and at -20 ºC as well.
Long-term Preservation
For long-term preservation, 500 μl of bacterial culture was grown in Trypticase Soy Broth (Oxoid,
England) at 37 ºC for 6 hours was taken in a sterile cryovial. Then 500 μl of sterile glycerol was added
to the broth culture and the cryovial was stored at -20 ºC.
Methods for Detection of Antibacterial Activity:
Preparation of Bacterial Suspensions
Using a sterile inoculating loop, one or two colonies of the organism to be tested were taken from the
subculture plate. The organism was suspended in 3 ml of physiological saline. The test tube containing
the saline was then vortexed to create an overall smooth suspension.
Comparing with the McFarland Solution
The bacterial suspension prepared was compared with the commercially available McFarland solution
2 (for detection of inhibition rate) and McFarland solution 0.5 (for detection of zone of inhibition by
agar disc/well diffusion method). A bacterial suspension which matches with McFarland 2 is supposed
to contain 6×10
8
colonies per ml. A bacterial suspension which matches with McFarland 0.5 is
supposed to contain 1.5×10
8
colonies per ml as described by McFarland (1907).
Comparative Study on the Antibacterial Activities of Neem Oil, Mustard oil and
Black Seed Oil Against Pathogenic Staphylococcus aureus, Klebsiella pneumoniae,
Salmonella Typhi and Pseudomonas Aeruginosa 61
61
Detection of Inhibition Rate
One hundred micro-litre of the both diluted and undiluted samples was spread on the agar plate
containing nutrient agar and from each diluted tube containing saline was spread immediately.
One hundred micro-litre from each diluted tube containing oils was spread on agar plate after
24 hour incubation.
CFU in saline and CFU in oil of each spread plate was counted and compared.
Rate of inhibition in case of every diluted tube was then calculated and averaged to detect
actual inhibition rate.
Test for Anti-Microbial Activity
Placement of the Oil Disc and Antibiotic Disc
Oil discs containing 20 µl concentration of Neem oil, Black seed oil and Mustard oil was made using
filter paper and then placed on the plates using a sterile forcep.
One sterile antibiotic disc was placed on the surface of an agar plate, using a forcep. The forcep
was sterilized by immersing the forceps in alcohol. It was then burnt. The discs were gently
pressed with the forcep to ensure complete contact with the agar surface. The disks were placed
away from the edge of the plates so that it is easily measured.
Once all disks are in place, the plates were inverted, and placed them in a 37 °C incubator for
24 hours.
Placement of Oil in the Well
Well was made in agar using a borer.
Twenty microlitre of oil was placed in the well using a pipette.
Zone of inhibition was then measured after 24 hours incubation at 37º C
Measuring Zone Sizes
Following incubation, the zone sizes were measured precisely using a ruler.
All measurements were made while viewing the back of the Petri-dish.
The zone size was recorded on the recording sheet.
Data Analysis
Data were analyzed using Microsoft excel version 2007.
Results
Clinical strain of the four bacterial species i.e. Staphylococcus aureus, Klebsiella pneumoniae,
Salmonella typhi and Pseudomonas aeruginosa, obtained from a tertiary hospital were streaked on the
respective selective media in order to determine and confirm the cultural properties of the organisms.
62 Sharmin Sultana, Nishat Anzum Shova, Akash Ahmed and M. Mahboob Hossain
Table 1: Biochemical test results of the target isolates.
Organisms
Biochemical tests
Indole
production
test
Methyl
red
reaction
test
VogesPro
skauer
reaction
test
Citrate
utilization
test
TSI Fermentation
Catalase
activity test
Oxidase
activity
test
Slant
Butt CO
2
H
2
S
Staphylococcus
aureus - + + + A A - - + -
Pseudomonas
aeruginosa - - - + K K - - + +
Klebsiella
pneumoniae - - + + A A + - + -
Salmonella typhi - + - - K A - + + -
KEY: A= acidic condition, K= alkaline condition, + = positive, - = negative
Comparison of the Growth of Organisms in Saline and Oils
Bacterial suspensions were taken and it was serially diluted in saline and in oils. Paraffin oil was not
used to dilute Neem, Black seed and Mustard oils because paraffin oil did not allow bacteria to grow.
Then same amount (100 µl) of diluted suspension was taken and spread on Nutrient agar. After
incubation oil treated suspensions showed fewer colonies appeared on the agar plate compared to the
non-treated suspension in physiological saline that of containing oils than saline which indicates that
these oils have antimicrobial activity against the selected pathogens. Number of colonies of the matted
plate and those plates which had more than 300 colonies were determined by back calculation.
Numbers of colonies in countable plates were calculated according to the formula given below;
CFU = Number of colonies × reciprocal of the dilution factor
volume of plated suspension
Figure 1:
Growth of Staphylococcus aureus after incubation with (a) Black oil at 10
-1
, (b) Neem seed oil at 10
-1
and (c)
Mustard oil at 10
-1
dilution of the suspension
(a) (b) (c)
Comparative Study on the Antibacterial Activities of Neem Oil, Mustard oil and
Black Seed Oil Against Pathogenic Staphylococcus aureus, Klebsiella pneumoniae,
Salmonella Typhi and Pseudomonas Aeruginosa 63
63
Determination of Inhibition Percentage
Number of colonies found in saline and oils suspension was compared for every dilution to find out the
rate of inhibition. Results of each set were then averaged to determine inhibition rate. Formula of
calculation of inhibition Percentage is:
େ୊୙ ୧୬ ୱୟ୪୧୬ୣ – େ୊୙ ୧୬ ୭୧୪
େ୊୙ ୧୬ ୱୟ୪୧୬ୣ
× 100
Table 2:
Total viable count of various bacteria in saline and in oils
Organism
Dilution of
the bacterial
suspensions
with oils and
saline
Saline
CFU/100
µl
Neem oil Black seed oil Mustard oil
CFU/100
µl
% of
inhibition
CFU/100
µl
% of
inhibition
CFU/100
µl
% of
inhibition
Staphyloc
occus
aureus
10
1
6.7 × 10
6
268 99.99 0 100 29.2 × 10
4
95.641
10
2
6.7 × 10
5
12 99.99 0 100 29.2 × 10
3
95.641
10
3
6.7 × 10
4
2 99.99 0 100 29.2 × 10
2
95.641
10
4
6.7 × 10
3
0 100 0 100 292 95.641
10
5
6.7 × 10
2
0 100 0 100 83 87.612
10
6
6.7 × 10
1
0 100 0 100 9 86.567
Klebsiella
pneumoni
ae
10
1
6.7 × 10
6
23 99.99 1.31 × 10
3
99.98 28.3 × 10
2
99.958
10
2
6.7 × 10
5
5 99.99 131 99.98 283 99.958
10
3
6.7 × 10
4
2 99.99 0 100 0 100
10
4
6.7 × 10
3
0 100 0 100 0 100
10
5
6.7 × 10
2
0 100 0 100 0 100
10
6
6.7 × 10
1
0 100 0 100 0 100
Pseudomo
nas
aeruginos
a
10
1
6.7 × 10
6
245 99.99 79 × 10
6
0 61 × 10
6
0
10
2
6.7 × 10
5
66 99.99 79 × 10
5
0 61 × 10
5
0
10
3
6.7 × 10
4
0 100 79 × 10
4
0 61 × 10
4
0
10
4
6.7 × 10
3
0 100 79 × 10
3
0 61 × 10
3
0
10
5
6.7 × 10
2
0 100 79 × 10
2
0 61 × 10
2
0
10
6
6.7 × 10
1
0 100 79 × 10
1
0 61 × 10
1
0
Salmonell
a typhi
10
1
6.1 × 10
6
1.86 × 10
4
99.69 1.83 ×10
3
99.97 23.9×10
2
99.96
10
2
6.1 × 10
5
1.86 × 10
3
99.69 183 99.97 239 99.96
10
3
6.1 × 10
4
186 99.69 0 100 25 99.95
10
4
6.1 × 10
3
49 99.19 0 100 4 99.93
10
5
6.1 × 10
2
2 99.67 0 100 0 100
10
6
6.1 × 10
1
0 100 0 100 0 100
Table 2 shows the percentages of inhibition of different bacteria by different oils at different
dilution of bacteria in saline and oil. Neem oil at 10
-1
dilution of bacteria inhibited the growth of
Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa and Salmonella typhi by
99.99%, 99.99%, 99.99%, 99.97% and 99.69% respectively. In same oil there was no growth from 10
-4
dilution except in case of Salmonella typhi and 100% inhibition of growth was observed at 10
-6
dilution. Black seed oil inhibited the growth of Staphylococcus aureus at all dilutions. In contrast the
same oil did not show inhibition of growth Pseudomonas aeruginosa at any dilution. Mustard Oil
inhibited the growth of Klebsiella Pneumoniae from dilution 10
-3
to 10
-6
but show inhibition 100% in
the same dilution. But in contrast the same oil did not show inhibition of growth Pseudomonas
aeruginosa at any dilution.
Selective antimicrobial activity test by means of antibiogram method
64 Sharmin Sultana, Nishat Anzum Shova, Akash Ahmed and M. Mahboob Hossain
All the five bacterial strains were subjected to the standard disc diffusion test with a control
antibiotic and paper disc and well containing oil. Control antibiotic for Staphylococcus aureus,
Klebsiella pneumoniae, Salmonella typhi was Chloramphenicol (30 µ g) and for Pseudomonas
aeruginosa was Cefepime (30 µg). The zone diameter of inhibition interprets the resistance and
sensitivity of the organisms to the respective antibiotics and oils. Presence of zone of inhibition around
oil disc or well containing oil means that these oils have antibacterial activity against the selected
pathogens.
Table 3: Zone of inhibition in response to oils and antibiotics
Organisms
Zone of inhibition (mm)
Antibiotic Neem oil Black seed oil Mustard oil
Well Disc Well Disc Well Disc
Staphylococcus aureus 26 17 9 43 31 0 0
Pseudomonas aeruginosa 29 0 0 0 0 0 0
Klebsiella pneumoniae 24 16 8 48 29 0 0
Salmonella typhi 22 18 8 13 12 0 0
Discussion
Plant essential oils and extracts have been used for many thousands of years, in food preservation,
pharmaceuticals, alternative medicine and natural therapies. Burt (2004) stated it is continuously used
as traditional treatment. These oils are available in Bangladesh and are potential sources of novel
antimicrobial compounds against selected bacterial pathogens. This in-vitro study showed that Neem
oil, Black seed oil and Mustard oil inhibited bacterial growth but their effectiveness varied.
In this study, inhibition rate of Neem, Black seed and Mustard oil was measured. Neem oil
exhibited more than 99% inhibition rate against the selected bacterial strains. Black seed oil exhibited
more than 99% inhibition rate against the selected bacterial strains except Pseudomonas aeruginosa.
Black seed oil did not have any antibacterial activity against Pseudomonas aeruginosa. Mustard oil
exhibited more than 93% inhibition rate against the selected bacterial strains except Pseudomonas
aeruginosa. Among all the oils tested in this work, Neem oil and Black seed were most effective as an
antibacterial agent.
In another study carried out by Tuhin Jahan, Zinnat Ara Begum and Sayeeda Sultana in 2017,
Neem oil was prepared by steam distillation process and its effect against S. typhi, E. coli and P.
aeruginosa was examined by detection of MIC by using ‘broth dilution method’ and by detection of
bacterial susceptibility by ‘Agar disc diffusion method.’ The MIC against S. aureus, S. typhi, E. coli
and P. aeruginosa was at 1:32, 1:16, 1:32 and 1:8 dilution. The average diameter of zone of inhibition
against S. aureus with neem oil was 19 mm whereas it was 30 mm with cefepime. S. typhi, E. coli and
P. aeruginosa exhibited zone of inhibition. Among all the test bacteria S. aureus had lowest MIC.
Jahan et al.,(2007) showed in vitro antibacterial activity of neem oil showed 92% susceptibility against
P. aeruginosa, S. pyogenes, E. coli, Proteus group and K. aerugenes. The MICs were varying between
¼ to 1/64 dilution. Inhibitory zones of 13-30 mm were obtained with 65.5% strains while 26.5% strains
showed zones of 8-12 mm.
An important characteristic of essential oils and their components is their hydrophobicity,
which enable them to partition the lipids of the bacterial cell membrane and mitochondria, disturbing
the cell structures and rendering them more permeable as described by Knobloch et al.,(1986).
Extensive leakage from bacterial cells or the exit of critical molecules and ions may lead to death. This
can be the reason of higher inhibition rate of oils.
A research done by Emeka LB, Emeka PM and Khan TM in 2015, evaluated the susceptibility
of multidrug resistant Staphylococcus aureus to Nigella sativa oil. Staphylococcus aureus was isolated
from 34 diabetic patient's wounds attending the Renaissance hospital, Nsukka, Southeast Nigeria. The
Comparative Study on the Antibacterial Activities of Neem Oil, Mustard oil and
Black Seed Oil Against Pathogenic Staphylococcus aureus, Klebsiella pneumoniae,
Salmonella Typhi and Pseudomonas Aeruginosa 65
65
isolates were characterized and identified using standard microbiological techniques. Isolates were
cultured and a comparative In vitro antibiotic susceptibility test was carried out using the disk diffusion
method. Of the 34 samples collected, 19 (56%) showed multidrug resistance to the commonly used
antibiotics. Nigella sativa oil was then studied for antibacterial activity against these multidrug
resistant isolates of Staphylococcus aureus in varying concentration by well diffusion method. Emeka
et al.,(2015) investigated black seed oil showed pronounced dose dependent antibacterial activity
against the isolates. Out of 19 isolates, 8 (42%) were sensitive to undiluted oil sample; 4 (21%) of these
showed sensitivity at 200 mg/ml, 400 mg/ml and 800 mg/ml respectively. Eleven (58%) of the isolates
were completely resistant to all the oil concentrations.
In the present study, the result of anti-bacterial activity of the oils by agar disc diffusion method
shown in table 3 revealed that Neem oil and Black seed oil possesses an effective antibacterial activity
against both gram positive and gram negative bacteria except Pseudomonas aeruginosa. In agar disc
diffusion test, Neem oil and Black seed oil showed zone of inhibition but Mustard oil did not show any
zone. Size of the zone of inhibition of Antibiotic disc was in between 22-29 mm. In most cases, Neem
oil gave smaller zone compared to antibiotic discs. On the contrary, Black seed oil gave larger zone of
inhibition than the control antibiotic disc (Chloramphenicol). None of the oils showed zone of
inhibition in case of Pseudomonas aeruginosa. According to Bakathir and Abbas (2011) the positive
inhibition of Black seed oil may be attributed to the two important active ingredients, Thymoquinone
and melanin. Neem oil also showed zone of inhibition. Mishra and Dave (2013) revealed it contains
active ingredients like azadirachtin, nimbin, picrin, and sialin. Mustard oil showed high inhibition rate
by dilution method but did not show any zone of inhibition against any organism. Mustard oil’s
viscosity can be a reason for this result. It is a possibility that the oil did not diffuse from the disc or
well into the agar. As a result, no zone of inhibition was found.
None of the oil exhibited zone of inhibition against Pseudomonas aeruginosa. Inhibition rate of
Black seed oil and Mustard oil was 0%. Though Neem oil showed more than 99% inhibition at dilution
method against Pseudomonas aeruginosa, no zone of inhibition was found. These results indicate that
Pseudomonas aeruginosa is not much sensitive to the oils tested.
Pseudomonas aeruginosa presents a great challenge in the clinical environment because of its
antibiotic resistance and prevalence of infection in patients with open wounds and compromised
immune systems. Its biofilms are difficult to destroy and its survival persists within and without its
host. Another ability of P. aeruginosa is to develop antibacterial resistance through mutational changes
in the function and production of chromosomally encoded resistance mechanisms. Furthermore, Lister
et al.,(2009) stated the most difficult challenge with this pathogen is the ability of P. aeruginosa to
become resistant during treatment of an infection.
The oils were not subjected to any dilution by using paraffin oil because in a separate test done
with only paraffin showed inhibition of growth. So, the results would not have been accurate using
paraffin as a diluent. According to a research, Paraffin is great source for storage of bacteria stock
culture but it is not suitable for bacterial growth because it has a strong inhibitory effect and weak
killing effect described by Hartsell (1953).
The demonstration of activity against other bacteria is an indication that the oil can be a source
of bioactive substances that could be of broad spectrum of activity. The fact that the oil was active
against Staphylococcus aureus and Klebsiella pneumoniae is also an indication that it can be a source
of very potent antibiotic substances that can be used against drug resistant microorganisms prevalent in
hospital environments.
Further studies are required to confirm this antibacterial activity and to separate the active
constituents and evaluate their antibacterial activity. It would be great if the exact antimicrobial
66 Sharmin Sultana, Nishat Anzum Shova, Akash Ahmed and M. Mahboob Hossain
compounds could be identified from the oils. Accordingly, the compounds need to be identified and
purified using high performance liquid chromatography (HPLC) or other high throughput technique.
Conclusion
From this study, it can be concluded that many essential oils possess antibacterial activity. Neem oil
and Black seed has the most potential bactericidal properties. The present investigation together with
previous studies provides support to the antibacterial properties of these oils. It can be used as
antibacterial supplement in the developing countries towards the development of new therapeutic
agents. Additional in vivo studies and clinical trials would be needed to justify and further evaluate the
potential of this oil as an antibacterial agent in topical or oral applications.
Conflict of Interest Disclosure
The authors declare that there is no conflict of interest regarding the publication of this paper.
Funding Acknowledgement
BRAC University
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... The results obtained from this study showed the predominant spectrum of black seed oil antibacterial action against B. subtilis and B. cereus, These results are supported by Javed et al [23] and Zuridah and his colleagues [24] they stated that the phenols and thymoquinone which are soluble in methanol and it reveals a great antibacterial effect against B. subtilis, Also a study by Shova, showed a similar result against B. cereus even at a very low concentration [25]. But Safhi et al. [26] disagreed with our results they showed negligible activity of N. sativa against B. subtilis. ...
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