Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College
of Veterinary Medicine. University of Basrah. Iraq
66
ANTIBACTERIAL ACTIVITY OF LAURUS NOBILUS EXTRACT
AGAINST Pseudomonas aeruginosa ISOLATED FROM WOUNDS IN
SHEEP AFTER FALSE WOOL SHEARIN
Nawres N. Jaber* , Nada S. Hadi* , Moaed H. Sayhood**
*Department of Veterinary Microbiology and Parasitology, College of Veterinary Medicine,
University of Basrah
2**Department of Veterinary Public Health, College of Veterinary Medicine, University of
Basrah
Corresponding A uthor: [email protected]
Keywords: false wool shearing , Pseudomonas aeruginosa, Laurus nobilus
ABSTRACT
Although sheep shearing is considered an important and a widely used process to cut off
the sheep’s wool, false wool shearing can cause serious problems by giving a chance to grow
bacteria. This study is aims to identify Pseudomonas aeruginosa isolated from the inflamed
wounds after false wool shearing process and to evaluate the antibacterial activity of Laurus
nobilus extract against this bacteria. The results of bacterial growth showed that P. aeruginosa
produced characteristic colonies on nutrient agar with pigment pyocin and β- hemolysis on blood
agar and grew on MacConkey agar but did not ferment lactose sugar. In addition, the isolates
were positive for biofilm formation using polystyrene 96 well plate. Among 6 antibiotic agents,
the highest resistance was found with novobiocin, chloramphenicol and tetracycline,
respectively. Laurus nobilis extract had an antimicrobial activity against P. aeruginosa. The
results of this study revealed that hot and cold alcoholic extracts of Laurus nobilis with MICs
6.5 mg/ml, 12.5 mg/ml and 50mg/ml, respectively, were more effective than hot water extract.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College
of Veterinary Medicine. University of Basrah. Iraq
67
INTRODUCTION
Pseudomonas aeruginosa (family Pseudomonadaceae) is an aerobic, motile, Gramnegative
rod, widely present in the environment, e.g. in water and in humid places (1), it is also
an important opportunistic pathogen for humans, plants and animals. P. aeruginosa can cause
acute and chronic infections in different mammalian hosts and organs due to the production of a
wide arsenal of virulence factors. Virulence factors associated with P. aeruginosa include
flagella, adhesion proteins and extracellular proteins, or secondary metabolites, with proteolytic
and/or cytotoxic activity (e.g. exotoxin A, elastase, proteases, pyocyanin, hemolysins) (2).
Inflammation of wounds is a defensive immune response that is coffered by the host against
foreign body (3). The innate immune system on encountering pathogen elicits the acute
inflammatory response that is accompanied by vascular leakage and leukocyte emigration. The
redness, swelling, heat and pain are natural signs of healing process during inflammation period
(4). The wound healing and tissue repair are complex processes that involve a series of
biochemical and cellular reaction (5). Most of the vascular changes observed in acute
inflammation are due to inflammatory mediators that are released by inflammatory cells at site of
wounds such as histamine and other mediators (3).
Laurus nobilis L, is one of the most well-known plants from the Lauraceae family, which
is also known as Bay or laurel leaves, it has an antimicrobial activity. Phytochemical studies on
Bay leaves and its fruits have indicated various secondary metabolites including alkaloids,
flavonols, glycosylated flavonols sesquiterpene lactones, monoterpene and germacrane alcohols
(6,7). Interestingly there is a worldwide concern around that use of antibiotic to treat bacterial
and fungal infection. Which can lead to rise and spread of organisms resistant to broad spectrum
antibiotic . That concern open ways to use plants as natural sources for novel antimicrobial
agents with a similar activity (8,9). Natural medicinal plants, as L. nobilis, are rich sources of
bioactive compounds. Thus, the biological properties of Bay extracts and its essential oil are
documented, specifically their antimicrobial activity.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College
of Veterinary Medicine. University of Basrah. Iraq
68
MATERIALS AND METHODS
Sample collection for isolation and identification of Pseudomonas aeruginosa: A total of 50
samples were collected from sheep wounds after wool shearing by using sterilized swabs. The
swabs were incubated in brain heart infusion broth for activation. Following incubation for 24
hrs. at 37◦C, the inoculums were streaked on nutrient agar for isolation, then the isolates were
cultured on blood agar and MacConkey agar and the plates were incubated overnight at 37◦C. P.
aeruginosa was identified by its colony characteristic, pigment production, grape like odor
formation. Suspected colonies were identified using motility test, Gram stain (10) and
biochemical test like catalase, oxidase, citrate utilization, methyl red test, Indole production and
growth at 42◦C (11).
Biofilm formation: Biofilm formation by P. aeruginosa was studied on 96 microtiter plate (12).
All the isolates were grown in trypticase soy broth with 0.25% sucrose and incubated overnight
at 37°C. Two hundred microliters were diluted overnight and the culture was transferred into 96-
well microtiter plate and incubated at 370C for 24 hrs., and the broth without culture was used as
a control. After incubation, the content of each well was gently removed by slightly tapping the
plates. The wells were then washed three times with 300 μl of sterile distilled water. Bacterial
adhering to the wells were fixed with 250 μl of methanol per well for 15 mins. Then each well of
plates was stained with 250 μl of 0.1% (w/v) crystal violet solution for 5 mins. Excess stain was
removed by washing with sterile distilled water and air dried.
Antibiotics susceptibility test: P. aeruginosa isolates that were inoculated on Mueller– Hinton
agar plates were incubated into nutrient broth overnight until the turbidity was equivalent to 0.5
Mcfarland standards, and then left for few minutes at room temperature. Antimicrobial
susceptibility was performed on Mueller-Hinton agar by the standard disk diffusion method. This
was done by dipping a sterile swab stick overnight in nutrient broth and the entire surface of
Mueller– Hinton agar plates was carefully swabbed. The antibiotics used against the tested
bacteria were: Tetracycline (10 μg), Ciprofloxacin (10 μg), Chloramphenicol (10 μg), ceftriaxone
(10 μg), Gentamicin (10 μg) and Novobiocin (10 μg). The antibiotic multi disc (Oxoid) was then
placed on the surface of the inoculated plates and gently pressed. The plates were incubated at
37◦C for 18–24 hrs. The diameter of the inhibition zone was measured in millimeters and the
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College
of Veterinary Medicine. University of Basrah. Iraq
69
isolates were scored as sensitive or resistant by comparing with the recommended values on
standard charts (13).
Preparation of Laurus nobilis extracts
Cold alcohol extract: Three hundred milliliter of 70 % ethyl alcohol were added to 50 gm of L.
nobilis leaves The mixture was stirred for 3 days at room temperature then filtered through
Whatmann No. 3. The filtrate was evaporated at 80°C by rotary evaporator, then poured in Petri
dish and left at room temperature to dry (14).
Hot water extract: Three hundred milliliters of distilled water were added to 50 gm of L. nobilis
leaves powder to obtain hot water extraction. The solution was left in reflux for 3 days then
filtered, and evaporated by rotary evaporator at 60°C (15).
Hot alcohol extract: Three hundred milliliter of 70 % ethanol were added to 50 gm of L. nobilis
powder to obtain hot alcohol extract. The solution was left in reflux for 3 days then filtered by
Whatmann No. 3 and evaporated by rotary evaporator at 60°C (15).
Antimicrobial activity:The antimicrobial activity was evaluated by the disk diffusion method
(16), which consists of using absorbent sterilized paper discs (9 mm in diameter) wetted with
extracts. The discs were placed on the surface of the agar. The bacteria were spread all over the
agar. The micro-organisms grew all over the surface of the agar except where the product that
would inhibit their growth. Following incubation around the discs, a clear circular inhibition
zone was observed. The effect of the extracts on P. aeruginosa was estimated by the appearance
of clear zones around the discs. The diameter of the halo of growth inhibition was measured by
vernier calipers (Mauser) and expressed in mm (including the diameter of the disc of 9 mm) (17).
All assays were performed in triplicate.
Determination of minimum inhibitory concentration (MIC): The minimum inhibitory
concentration (MIC) of L. nobilis extracts was determined by using disc diffusion method
according to (18) with a little modification. Briefly, sterilized filter paper discs (9 mm in
diameter) were impregnated with different concentrations of L. nobilis extracts (50 mg/ml, 25
mg/ml, 12.5 mg/ml and 6.25 mg/ml), which were prepared before 24 hrs and kept at 4◦C. Muller
Hinton agar media were seeded with 0.1 ml of bacterial culture and adjusted to 0.5 McFarland
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College
of Veterinary Medicine. University of Basrah. Iraq
turbidity standard. After 15 min
agar. MIC was defined as the lowest concentration that inhibit the visible bacterial growth.
The gross sections in Fig
days of infection, respectively,
aeruginosa were isolated by using nutrient agar which were confirmed primarily based on
characteristic colony morphology in
Gram’s staining technique. P
odor and produced characteristic pigment pyocins in nutrient agar
β-hemolysis on blood agar (Fig
(Fig. 5). Thin smears were prepared on glass slides from a single colony for Gram’s staining.
Following staining, the morphology of
shaped appearance. The motility test revealed that
purified isolates of P. aeruginosa
Fig. 1: Contaminated wound after three
70
fter min, the prepared discs were placed on the surface of
RESULTS
Fig. 1 and 2 showed the contaminated wound
, following false wool shearing process.
nutrient agar, blood agar and MacConkey agar media and
P. aeruginosa produced mucoid colonies with emitted
(Fig. 3). The isolates produced
Fig. 4) and on MacConkey agar, but did not ferment lactose sugar
the isolated P. aeruginosa showed Gram
. the isolates were found to be motile.
were examined by different biochemical reactions
ontaminated days from infection period
the seeded
wounds after three and five
Ten isolates of P.
d sweat grape
). ot Gram-negative, rod
The
(Table 1).
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College
of Veterinary Medicine. University of Basrah. Iraq
Fig. 2: Contaminated wound after five days from infection period with ab
71
ontaminated abscess appearance.
cess
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College
of Veterinary Medicine. University of Basrah. Iraq
Fig. 3: Pyocin production on
nutrient agar by
72
P. aeruginosa
Fig. 4: β- hemolysis on blood agar
by P. aeruginosa
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College
of Veterinary Medicine. University of Basrah. Iraq
Fig.
Table 1
biochemical reactions
Oxidase
Catalase
Indol production
Citrate utilization
Methyl red test
growth at 42C
Biofilm formation
Biofilm formation by P. aeruginosa
bottom tissue culture plates at 37
were positive for biofilm formation
73
5: Bacterial growth on MacConkey agar
1: Biochemical test of P. aeruginosa
Result
xidase +
+
-
+
+
+
was also studied on polystyrene, 96 well
ºC for 24 hrs. It was found that all t
(Fig. 6).
well-flat
the isolates
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College
of Veterinary Medicine. University of Basrah. Iraq
74
Fig. 6: Biofilm production by P. aeruginosa in microtiter plate
Antibiotics sensitivity test
Antimicrobial sensitivity test was done on 6 antimicrobial agents using:
Tetracycline (10 μg), Ciprofloxacin (10 μg), Chloramphenicol (10 μg), ceftriaxone
(10 μg) , Gentamicin (10 μg) and Novobiocin (10 μg), (Table 2) .
Table 2: Antibiotics sensitivity test results
Antibiotics Symbol Disc content
( mcg )
Diameter of inhibition (mm )
Sensitive intermediate Resistance
Tetracycline TE 10 - - ± 1.3 0≤ 14
Ciprofloxacin CIP 10 ±30.3 ≥21 -
Chloramphenicol C 10 - - ± 2.33≤ 12
Ceftriaxone CRO 10 - - 0 ≤
Gentamicin CN 10 ±24.33 ≥ 15 - -
Novobiocin NV 10 - - ± 1.66 ≤ 18
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College
of Veterinary Medicine. University of Basrah. Iraq
Antimicrobial activity of Laurus nobilis
Following the measure of
extract and cold alcohol extract were more effective than ho
Table 3: Antimicrobial activity of
Mean and standard deviation of Inhibition zone diameter
Hot alcohol extract (
100mg/ml )
33±0.75
Fig. 7: Antimicrobial activity of
Determination of minimum inhibitory concentration (MIC)
Hot ethanolic extract of
mg/ml against P. aeruginosa.
extract showed less activity against
75
extracts
the antibacterial activity of L. nobilis extracts,
old hot water extract
L. nobilis extracts
Cold alcohol extract(
100mg/ml )
water extract
23±0.23
L. nobilis against P. aeruginosa
L. nobilis had the highest bacterial activity with an MICs of
. Cold ethanolic extract had an MICs of 12.5 mg/ml
P. aeruginosa with an MICs of 50 mg/ml
1
3
2
the hot alcohol
(Fig. 7) and table 3.
extract( 100mg/ml )
19±0.23
6.5
ml, while the water
(Fig. 8 )
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College
of Veterinary Medicine. University of Basrah. Iraq
Fig. 8: M
Pseudomonas aeruginosa
to the Pseudomonadaceae family
vegetation and water, marshes, and coastal marine habitats (
humans.
P. aeruginosa has been known to cause opportunistic and chronic infections in wounds
where it produces series of virulence factors such as enzymes and toxins which breakdown the
host’s tissues (20). Mucus and extracellular polymeric substances including alginate
produced by the organism to prevent the action of phagocytes and other immune mechanisms
(21). The ability to multiply quickly and produce biofilm within a short period (
its pathogenicity. P. aeruginosa
(23). The ability to form biofilm enhances the resistance to the
antimicrobials, which further increases the virulence capability this phenomenon has been linked
to the impediment of wound healing and cause
The present study exhibited the medical importance of
antimicrobial activity of cold
Mean of inhibition of growth
76
Minimum inhibitory concentration (MIC)
DISCUSSION
is a Gram-negative, aerobic rod shaped bacterium that belongs
family. It is a free-living organism, commonly found in soil,
19) and occasionally animals and
). ). is one of the organisms that are predominant in wound infections
. body’s
causes of chronic wounds (24).
resent the plant through
and hot alcoholic extracts and water extract against
) are often
22) has enhanced
hat s immune system and
the existence of
P. aeruginosa
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College
of Veterinary Medicine. University of Basrah. Iraq
77
isolated from the inflammatory wound after false wool shearing in sheep .The results showed
that cold and hot alcoholic extracts were more effective than hot water extract. The average
diameter of inhibition zone ranges from 33 ± 0.75 , 23 ± 0.23 and 19 ± 0.23 respectively. The
antimicrobial activity depends on the antimicrobial compound in the extracts according to the
solvent used in the process (25). The hot extract showed the lowest MICs comparing to the other
types of extracts and this may be due to the large quantity of active substance that were
precipitated during the extraction process due to the solvent.
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