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ANTIBACTERIAL EFFICACY OF ETHANOLIC EXTRACT OF GARLIC
(Allium sativum) AND SUMAC (Rhus coriaria) AND ANTIBIOTIC ON
Salmonella typhimurium ISOLATED FROM CHICKENS
Mahasen A.Khudair *, Jenan Nadhim Sadeq ** , Zahira A. AL-Zuhairi***
*Department of pathology and Poultry diseases, College of Veterinary Medicine, University of
Al-Qadisiyiah, Al-Qadisiyiah,Iraq.
**Department of Microbiology and Parasitology, College of Veterinary Medicine, University of
Al-Qadisiyiah, Al-Qadisiyiah,Iraq.
***Department of Public Health College of Veterinary Medicine, University of Al-Qadisiyiah,
Al-Qadisiyiah,Iraq.
Corresponding Author: [email protected]
Keywords: Garlic extract, S. typhimurium, Sumac extract.
ABSTRACT
Antibacterial drug resistance is an increasingly worldwide occurred health problem
presented by bacterial-originated defectiveness to the work of a wide-range of antibacterial
drugs. Uncovering the antibacterial effects of ethanolic extract of garlic (Allium sativum) (Glc)
and sumac (Rhus coriaria) (Smc) on Salmonella typhimurium isolated from chickens was the
main goal of the present study.Fifty samples of intestinal contents of chickens were collected
randomly from various farms located in Al-Diwaniyah province,All specimens inoculated into
on macconky agar, Salmonella-Shigella agar at 37c for 24-48-hr, also examined on XLD agar
and Salmonella CHROME agar Allium sativum( Glc) or Rhus coriaria (Smc )extract, at
different concentrations, or antibacterial drugs (control), 10mcg ciprofloxacin (Cip), 30mcg
amoxicillin/clavulanic acid (Amc), 10mcg neomycin (N), were employed to test their
antibacterial activities (AAs) against S. typhimurium using agar-gel diffusion tests, The
experiment included an investigation about of one isolates from origin 6 isolates of S.
typhimurium , 6 out of the 50 chicken samples (12%) were culture positive for salmonella
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typhimurium , Significant (p˂0.05) increases in the AAs against S. typhimurium were shown by
Glc or Smc extracts when compared to those from the antibiotics. Moreover, these AA increases
were revealed to be incremented as the concentrations of those extracts were elevated. No
significant (p˃0.05) differences were demonstrated between the AAs of both extracts. In
addition, Cip, Amc, and N showed AAs against S. typhimurium; however, Cip revealed the
strongest AAs followed by Amc.
INTRODUCTION
Resistance to antibiotics is a historic and a diverse health issue. Overcrowding, elevated
world moving of people, leveled up of utilization of antibiotics in hospitals and food animal
industries, selection pressure, low levels of hygiene, non-domestic life spreading, and improper
waste disposal are some of the main factors that play important roles in dissemination of this
problem. The medical intervention using antibiotics is one of the most important approaches in
modern medicine to the fight against infectious disease (1, 2, 3). The "golden age" of the
discovery of antibiotics was between the nineteen and sixties of the previous century resulted in
finding numerous novel antibiotics. Unfortunately, that age came to an end, since scientists could
not catch up with antibiotic discovery challenged by the presence of evolved pathogenic
microorganisms with antibacterial resistance properties. The predisposing factors connected to
the development of antibiotic resistance are the constant inability to create or find novel
antibiotics and misuse of those drugs (4).
Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa are
currently among the most famous antibiotic-resistant bacteria. Multidrug Resistant (MDR)
microorganisms are categorized as three distinct (i.e. urgent, serious and concerning) levels in
the United States by the Centers for Disease Control and Prevention (CDC). Resistance against a
particular antibiotic is comparative to the examined microorganisms and their prior exposures to
antibiotics. Intrinsic and obtained are two different kinds of antibiotic resistance. As a
consequence of vertically transmitted genetic materials, microorganisms may be intrinsically
insensitive to some antibiotics. A specific antibiotic, for example, can not completely enter the
outside layer of some microorganisms, or efflux pumps can remove the antibiotic which enters
the membrane (5, 6, 7, 8).
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The modern integrative healthcare (IHC) facilities are becoming progressively included
with complementary and alternative medicine (CAM). Scholars have progressively reported that
CAM is common and / or efficient in the integration of healthcare literature to treat and manage
chronic illnesses, and particularly discomfort and stress-related diseases. CAM may offer
possibly more efficient multidisciplinary solutions to the treatment of complicated chronic
illnesses like HIV, acute ache and addiction. CAM may include the use of certain protocols or
substances such as natural products, plants, and/or plant extracts (3, 9, 10, 11, 12).
Uncovering the antibacterial effects of ethanolic extract of garlic (Allium sativum) (Glc)
and sumac (Rhus coriaria) (Smc) on Salmonella typhimurium isolated from poultry was the main
goal of the present study.
MATERIALS AND METHODS
Intestinal samples and bacterial isolation
Fifty samples of intestinal contents of chickens were collected randomly from various
farms located in Al-Diwaniyah province, Iraq. Samples were processed in the laboratory of
Poultry Disease College of Veterinary Medicine, University of Al-Qadisiyah for bacteriological
assay. Each sample was inoculated in 5ml of nutrient broth and incubated at 37°C for 24-48hrs.
Then, by using a sterile loop, some of the cultivation from the broth was collected and streaked
on MacConky agar. Visual examination of the bacterial colonies and Gram staining were
produced. The samples were subculture and examined on xylose lysine deoxycholate (XLD)
agar, then incubated for 24 hours at 37˚C. The isolates were activated by inoculating some of the
colonies onto Salmonella Chrome agar (SCA) and incubated at 37˚C overnight. In addition,
Vitek2system was employed for rapid identification of the isolates.
Plants and extraction
Bulbs of garlic and sumac seeds were bought from local stores in Al-Diwaniyah City.
The Bulbs were washed, sun-dried out, and sliced. Then, the sumac seeds and the dry garlic
slices were grinded separately to produce a powder-like form and stored in nylon bags until use.
Ethanolic-based extractions were conducted according to (13). Briefly, 50gm of each powder
was mixed up with 250ml of 96% ethanol. The combination was maintained at room temperature
for 2-5 days in closed bottles and rocked many times per a day. A filtering paper was used. The
rest was extracted 3 to 5 times until a definite supernatant had been acquired. 40oC rotary
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evaporator was used. Weighing and storing the finished products at-20oC until use. The yield
percentages of sumac (Rhus coriaria) 14.12% while to allium sativum was 9.25% (13).
Antibacterial susceptibility test
A serial dilution of diluting 2gm of each extract with 30% ethanol at 5ml resulting in a
400mg/ml stock solution. Working concentrations of 200, 100, 50, and 25mg/ml (each was done
by mixing 1ml of 30% ethanol with 1ml of the extract solution) were generated.
Muller Hinton media (MHM) were used for the well diffusion method (14). In the test,
the suspension of bacterial growth was moved over the entire surface of each plate by using
cotton swabs excluding six uniform wells of 5mm diameters (15). A sterile cotton swab was
employed dipping into the suspension of bacterial growth and inoculated onto the MHM surface
by streaking swabs which was used for the antibiotic study, After drying the inoculums, 0.1ml
each extract’s concentration was placed into the wells besides 0.1ml of 30% ethanol, a negative
control, on the same extract plate. Then, the petri-dishes were left for 4-6hrs for the plant extract
to spread over in the cultivating medium. Antibacterial drugs (control), 10mcg ciprofloxacin
(Cip), 30mcg amoxicillin/clavulanic acid (Amc), 10mcg neomycin (N), were employed to test
their antibacterial activities (AAs) against S. typhimurium using agar-gel diffusion tests on
different plates. These experiments were done in triplicates for each extract and for each
antibacterial drug. Then, all treatments were placed in an incubator at 37ºC for 24hrs. Following
that, the zone of inhibition’s diameter was measured in millimeters by using a ruler (16).
Statistical analysis
Mean ±SE was considered, ANOVA test, least significant differences (LSD) were
employed, and pRESULTS
Bacterial isolation: Out of 50 intestinal samples , 8 were suspect Salmonella spp isolates, All
the suspect S spp isolates were colorless and transparent colonies on MacConkey agar media
(figure: 1) S. spp circular colonies of pale color and a black centers on the XLD agar (figure: 2)
while characterized by rounded pink colonies on salmonella CHROME agar media (figure: 3)
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding o
Veterinary Medicine. University of Basrah. Iraq
Figure 1; suspect S. spp colorless and
transparent colonies on MacConkey agar.
Figure 3; suspect S
of the 17th International
Iraq.
252
c o l o r l e s s a n d
S. spp rounded pink colonies on the salmonella CHROM
agar.
Figure 2; suspect S. spp circular
transparent colonies and black centers on
f Conference. College of
and
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From 8 suspected isolates Six has been confirmed as typhimurium isolates were confirmed
by vitek 2 ,according to vitek2 Technique result (64 biochemical tests) ,out 50 sample were
typhimurium prevalence rate in intestinal samples was (12%) .
Antibacterial activity: Significant (p˂0.05) increases in the AAs against S. typhimurium were
shown by Glc or Smc extracts when compared to those from the control drugs. Moreover, these
AA increases were revealed to be incremented as the concentrations of those extracts were
elevated, table 1 and 2 and figures 4-8. No significant (p>0.05) differences were demonstrated
between the AAs of both extracts. In addition, Cip, Amc, and N showed AAs against S.
typhimurium; however, Cip revealed the strongest AAs followed by Amc. The results showed
there are significant difference(p˂0.05) between the used antibiotic & Garlic extract which give
the highest zone of inhibition (30.3±0.68) mm.
Also, the Sumac extract showed significant increasing (p˂0.05) in inhibition zone
(29.76±0.959) mm as compared with other used antibiotics.
Table (1): Inhibition zones (mm) of S. typhimurium growth by extracts of sumac and garlic in
culture media.
Extract concentration (mg/ml)
Type of plant 25 50 100 200 400
29.7±0.95
Ae
26.4±0.86
Ad
23.2±0.75
Ac
20.4±0.29
Ab
17.9±1.20
Aa
Sumac
30.3±0.68
Ac
24.5±2.05
Ab
22.7±1.27
Ab
22.5±0.66
Ab
18.6±0.77
Aa
Garlic
Different lowercase letters mean significance for horizontal comparisons.
Different uppercase letters mean significance for vertical comparisons.
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Table (2): Inhibition zones (mm) of S. typhimurium growth by antibacterial drugs, control, in
culture media
Positive and negative control Inhibition zone (mm)
Ciprofloxacin (Cip) 24.8±0.13a
Amoxicillin/Clavulanic acid (Amc) 20.9±0.66b
Neomycin(N) 11.3±0.33c
Different lowercase letters mean significance difference (P<0.05)
Table (3): Inhibition zones (mm) of S. typhimurium growth by extracts of garlic, sumac and
antibacterial drugs in culture media.
Plant extract and antibiotic Inhibition zone(mm)
Garlic 30.3±0.68a
sumac 29.76±0.959a
Neomycin(N) 11.33±0.333b
Amoxicillin/Clavulanic acid (Amc) 20.96±0.66c
Ciprofloxacin (Cip) 24.86±0.133d
Diluted ethanol (30%) 0±0e
Different lowercase letters mean significance difference (P<0.05)
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Figure 4; Inhibition zones produced by sumac (Rhus
coraria) against S. typhimurium.
Figure 5; Inhibition zones produced by sumac (Rhus
coraria) against S. typhimurium.
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Figure 6; Inhibition zones produced by garlic (Allium sativum) against S. typhimurium.
Figure 7; Inhibition zones produced by garlic (Allium sativum) against S. typhimurium.
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DISCUSSION
Since the clinical implementation in the 1940s, problems linked to the use of antibiotics
have been recognized. The use and often improper use of those agents have since increased.
Antibiotics are strong medicines used to fight deadly infectious illnesses. Antibiotics have a
broad variety of harmful impacts like all strong medicines. An elevated consuming impact that
neglects the danger of this use leads to diminish the beneficial effects of those agents. However,
when antibiotics are appropriately used, there provide advantages for patients as long as they
adhere to the consuming rules. In addition, the structure of the pathogenic microorganism is
destructed by those drugs, contributing to bacterial adjustment or mutations, encouraging the
production of new resistant strains to those antibiotics (19).
The present work unveiled the antibacterial effects of sumac on S. typhimurium. The actions
of herbal extracts, like thyme, garlic, rosemary, sumac, spice, ginger and mustard, have been
earlier explored as antibacterial and antioxidizing inhibitors. The antimicrobial impacts on
foodborne bacteria of Smc have been demonstrated. Tannins and other compounds are the main
reason for the antibacterial activity of sumac. Moreover, the elevated concentrations of carvacrol
Figure 8; Inhibition zones produced by the antibacterial drugs, 10mcg ciprofloxacin (Cip),
30mcg amoxicillin/clavulanic acid (Amc), 10mcg neomycin (N), against S. typhimurium.
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and thymol in Smc are associated with antioxidant and AAs in Smc (20-23)The AAs of Smc
against Pseudomonas spp., Enterobacteriaceae, lactic acid bacteria, and yeast-mold of meat
were tested and revealed to be strong in the low concentration, negative correlation with
concentration of Smc, (24) however, this disagrees with the present study data that showed a
positive correlation with concentration.
The current results demonstrated the antibacterial effects of garlic on S. typhimurium.
Alcohol extract was discovered to be much more durable and efficient than aqueous extract.
Tests have shown, however, that the AA of garlic depends entirely on the allicin molecule, which
is extra efficient on gram-positive bacteria. Although more lipid materials in the membranes of S.
typhimurium are present making allicin unable to achieve this objective, as it is caught in this
lipid material (25).
The present study data showed strong effects of garlic against the growth of this bacterium.
Antimicrobial characteristics for garlic have been shown to have a range of anions such as
nitrate, sulfide, chloride, and organosulfur substances. Furthermore, many study findings have
shown that allicin is the most significant antibacterial drug, reducing RNA production and
capturing RNA (25). The data of the present study show strong AAs of the Glc or Smc against S.
typhimurium. The present work also recorded the significant (P<0.05) elevation of antibacterial
activity of the both plant extract (garlic and sumac) versus the activity of tested antibiotics
(neomycin, amoxicillin, ciprofloxacin) and this results may be attributed to the resistance of the
isolated bacteria toward the above antibiotics due to misuse of these drugs in clinical fields
which lead to restriction in the antibiotic activity against tested bacteria (26).
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