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1 FigureInhibitory effect of aqueous garlic (Allium sativum) extract against clinical isolates of Salmonella typhi
Abstract
Typhoid
Fever is a systemic disease which is more prevalent in under-developed
countries. Drug resistance has been developed against antibiotics used
for the treatment of typhoid. It is very important to set the basis of
alternative medicines for management of typhoid. Garlic is one of the
natural plants being used as spicy food and folk medicine. Various
beneficial therapeutic effects of garlic have been documented including
anti-microbial, hypolipodemic and anti-oxidant. The objective of present
study was to explore anti-bacterial activity of aqueous garlic extract
against Salmonella typhi. A total of 50 clinical isolates of S. typhi
including 30 multi-drug resistant (MDR) and 20 antibiotic sensitive
isolates were investigated to check the inhibitory effects of garlic
extract; screening was done by agar well diffusion assay and minimal
inhibitory concentration (MIC) was performed by agar dilution technique.
MIC of garlic extract ranged from 18-22 mg/ml; showing the inhibitory
activity of garlic extract against S. typhi isolates. It is worth
describing that garlic might be utilized as anti-typhoid agent after
determining its pharmacokinetics and pharmacodynamics.
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Available from: Muhammad Usman Qamar
African Journal of Microbiology Research Vol. 6(21), pp. 4475-4480, 9 June, 2012
Available online at http://www.academicjournals.org/AJMR
DOI: 10.5897/AJMR11.540
ISSN 1996-0808 ©2012 Academic Journals
Full Length Research Paper
Inhibitory effect of aqueous garlic (Allium sativum)
extract against clinical isolates of Salmonella typhi
Abdul Hannan1, Kanwal Rauf1, Muhammad Ikram Ullah2, Tahir Naeem3, Mehwish Raja1,
Muhammad Usman Qamar1, Romeeza Tahir4 and Mehwish Saba1
1Department of Microbiology, University of Health Sciences, Lahore-Pakistan.
2Department of Biochemistry, University of Health Sciences, Lahore-Pakistan.
3Bacteriology, King Saud Hospital, Kingdom of Saudi Arabia.
4Department of Immunology, University of Health Sciences, Lahore-Pakistan.
Accepted 6 February, 2012
Typhoid Fever is a systemic disease which is more prevalent in under-developed countries. Drug
resistance has been developed against antibiotics used for the treatment of typhoid. It is very important
to set the basis of alternative medicines for management of typhoid. Garlic is one of the natural plants
being used as spicy food and folk medicine. Various beneficial therapeutic effects of garlic have been
documented including anti-microbial, hypolipodemic and anti-oxidant. The objective of present study
was to explore anti-bacterial activity of aqueous garlic extract against Salmonella typhi. A total of 50
clinical isolates of S. typhi including 30 multi-drug resistant (MDR) and 20 antibiotic sensitive isolates
were investigated to check the inhibitory effects of garlic extract; screening was done by agar well
diffusion assay and minimal inhibitory concentration (MIC) was performed by agar dilution technique.
MIC of garlic extract ranged from 18-22 mg/ml; showing the inhibitory activity of garlic extract against S.
typhi isolates. It is worth describing that garlic might be utilized as anti-typhoid agent after determining
its pharmacokinetics and pharmacodynamics.
Key words: Antibacterial activity, garlic extract, Salmonella typhi, minimal inhibitory concentration (MIC), agar
dilution.
INTRODUCTION
Typhoid fever still causes substantial illness and deaths
in many parts of the world, especially in developing
nations (Ochiai et al., 2005). According to a report
published WHO bulletin in 2000, the estimated global
incidence of typhoid fever was about 21.6 million and
mortality rate was up to 216,000 per year. The highest
incidence rate of typhoid fever (>100/100,000 cases/year)
has been reported in south-central Asia and south-east
Asia (Crump et al., 2004). In 2008, it was reported that
the incidence of typhoid fever is 451/100,000 in Pakistan
(Kothari et al., 2008).
Multi drug resistant (MDR) Salmonella typhi showed
resistance to all three first line drugs {Ampicillin (AMP),
Chloramphenicol (C), and Trimethoprim-sulfamethoxazole
*Corresponding author. E-mail: kanwal.rauf@gmail.com. Tel:
+92334-4153248.
(SXT)} (Ackers et al., 2000). MDR S. typhi emerged
somewhere in the mid 1990s and are now reported in
different regions of the world (Ward and Thelfall, 2001;
Parry et al., 2002). Under these circumstances, third
generation cephalosporin namely, ceftriaxone (CRO)
appears to be the most reliable choice for MDR and
nalidixic acid resistant isolates of S. typhi (NARST)
(Bhutta, 2006). In 1999, high level resistance to CRO
(MIC 64 mg/L) has been reported from Bangladesh
(Parry et al., 2002; Saha et al., 1999). Recently extended
spectrum beta lactamase (ESBL) production has been
reported in S. typhi isolate isolated from the 54 year old
Dutch man returned from Philippines (Naiemi et al.,
2008).
This continuous spread of MDR pathogens and cost-
effectiveness of drug regimen has become a serious
threat to public health and infection control practitioners.
The multiple and repeated difficulties with antibiotics has
prompted research to explore alternate agents.
4476 Afr. J. Microbiol. Res.
Phytotherapeutic agents like garlic (Allium sativum)
which is frequently used in alternative medicine has
gained immense interest in medical literature (Iwalokun et
al., 2004). Recently, garlic has been found to be an
effective agent for its application as anti-tumor, anti-
oxidant, anti-viral, anti-fungal, anti-microbial, anti-
thrombotic, anti-inflammatory, hypoglycemic, immune
modulatory effects (Amagase et al., 2001).
Garlic holds up the highest concentration of sulfur
compounds like allicin and others having biological
activities. These are responsible for not only its pungent
smell but also for its medicinal value (Kemper, 2000). The
thiosulfate allicin has effective anti-microbial properties
and other non-sulphur constituents like proteins,
saponins and phenolic compounds may also contribute to
its anti-microbial activity (Corzo-Martinez et al., 2007).
Garlic is present in various forms namely, raw juice,
garlic oil, garlic powder, and many types of extracts like,
aged garlic extract, aqueous garlic extract (AGE),
methanolic extract, ethanolic extract and many more
(Jabar and Al-Mossawi, 2007). Different garlic extract
preparations demonstrated their in-vitro activity against
Gram-negative and Gram-positive bacteria including
species of Escherichia, Staphylococcus, Streptococcus,
Klebsiella, Proteus, Bacillus, Clostridium and even acid-
fast bacilli (AFB) such as Mycobacterium tuberculosis
(MTB) (Uchida et al., 1975). It is also effective against
antibiotic resistant isolates like methicillin-resistant
Staphylococcus aureus (MRSA) as well as other MDR
enterotoxicogenic isolates of Escherichia coli (ETEC),
Salmonella goldcoast and Klebsiellae (Jabar and Al-
Mossawi, 2007) and also MDR MTB (Hannan et al.,
2011).
Garlic having multiple biological properties; inspired to
investigate local cultivated garlic (small cloves) for its
anti-typhoid effects against sensitive and MDR S. typhi
isolates which might be helpful in combating this public
health concern issue.
MATERIALS AND METHODS
Prior to the start of the study, approval was obtained from the
Ethical Committee, University of Health Sciences, Lahore, Pakistan.
Bacterial isolates
Fifty clinical isolates of S. typhi were provided by the Department of
Microbiology, Armed Force Institute of Pathology, Rawalpindi
(AFIP), Army Medical College Rawalpindi, Pakistan (AMC) and
Microbiology Department of Sheikh Zayed Medical Complex,
Lahore (SZH).
Confirmation of isolates
These isolates were reconfirmed by various microbiological
techniques namely, Gram stain, catalase test oxidase test and API
20E. Antibiotic profile has been performed by Kirby-Bauer disk
diffusion method and the interpretation of the susceptibility was
done according to interpretation break point, given in Clinical
Laboratory Standards Institute (CLSI) 2009 guidelines (Wikler et al.,
2009).
Aqueous garlic extract (AGE) preparation
Fresh garlic was purchased from local market. The aqueous garlic
extract was prepared according to “Iwalokun BA” method. The
cloves were separated and peeled to obtain the edible portion. Fifty
Gram of the edible portion was chopped and homogenized in 100
ml autoclaved distilled water in a blender. The homogenate was
filtered through 25 µm pore-size (Millipore) filter paper (Iwalokun et
al., 2004).
Storage of extract stock solution
The stock concentration of 50 g/100 ml or 500 mg/ml of garlic
extract was obtained and stored at 4°C in sterile screw cap vial (50
ml).
Anti-bacterial testing
The extract was screened against an isolate of MDR S. typhi,
(AMC-3) by agar well diffusion assay. Staphylococcus aureus,
ATCC (25923) and S. typhimurium, ATCC (14028) were used as
quality control. The subculture isolates were adjusted to 0.5
McFarland standard and lawned on Mueller Hinton (MH) agar. Four
serial dilutions (1/2; 250 mg/ml, 1/4; 125 mg/ml, 1/8; 62.5 mg/ml,
1/16; 31.25 mg/ml) were prepared from the stock solution of
aqueous garlic extract in normal saline. Then stock solution of
extract and its serial dilutions were filled in precut wells made in
agar with 6 mm diameter sterile steel borer. The plates were
incubated for overnight at 35-37°C. Bacteria showing a clear zone
of more than 12 mm were considered to be inhibited. Two controls
were run simultaneously, distilled water (diluent) was used as
negative control and Ciprofloxacin (5 μg) serve as positive control.
This procedure was performed in duplicate.
Determination of Minimal Inhibitory concentration (MIC)
MIC was determined by agar dilution method. AGE was mixed with
sterilize MH agar at 50°C to achieve the desired concentrations
from 0.5 to 30 mg/ml. The grid was filled with 500 µl of 0.5
McFarland standard bacterial suspensions. Then it was placed side
by side with extract incorporated plates in a multi-inoculator. All
plates were punched with 3 µl of bacterial suspensions and
simultaneously 35 isolates were inoculated at single plate including
the ATCC strains. Then the plates were incubated for over night at
appropriate temperature. Two control plates were also set up in
parallel. The positive control contains MH agar plate without extract
and inoculated with all isolates to confirm the viability of the cultures
while the sterility control contains un-inoculated MH. The lowest
concentration of AGE at which all bacterial isolates were inhibited
was MIC.
Data analysis
Data was analyzed using Statistical Package for Social Sciences
(SPSS) version 17.0.
RESULTS
According to the anti-microbial sensitivity profile results,
Table 1. Anti-microbial susceptibility pattern of S. typhi to first-line
anti-typhoid drugs.
S. typhi isolates
No (%)
MDR isolates*
30 (60)
Sensitive isolates
20 (40)
Total
50 (100)
*MDR; Multi-drug resistant.
out of 50 S. typhi isolates 30 isolates (60%) were MDR
S. typhi while 20 (40%) were sensitive to all three first line
drugs namely C, SXT and AMP (Table 1).
Anti-bacterial activity of AGE
The sizes of inhibition zones were inversely proportional
to the increase in dilution of AGE. The most sensitive
strain was ATCC S. typhimurium (14028) which showed
the largest zone (16±1 mm) of inhibition at the highest
dilution (1/16; 31.25 mg/ml), followed by MDR S. typhi
(12.5±1.5 mm). The AGE did not exhibit any zone against
ATCC S. aureus (25923) at the same dilution. MDR S.
typhi isolate showed zone of inhibition >12 mm at all
serial dilutions except 1/16 (31.25 mg/ml). The results are
given in Table 2 and Figure 1.
MIC of AGE
Maximum numbers of sensitive isolates (n=9) were
inhibited at 18 mg/ml while MDR S. typhi isolates (n=9) at
22 mg/ml (Table 3). Table 4 represents MIC range of
garlic extract (18-22 mg/ml) on 50 isolates of S. typhi.
MIC100 for sensitive and MDR isolates was same as 22
mg/ml while MIC90 for sensitive and MDR isolates was >
20 and > 21 mg/ml respectively. Figure 2 showed the
cumulative percentage of MDR S. typhi which is 13, 30,
50, 70 and 100% at concentrations of 18, 19, 20, 21 and
22 mg/ml of garlic extract respectively.
DISCUSSION
The widespread and injudicious use of antimicrobial
agents in human has led to the development of extensive
resistance amongst bacterial pathogens. Bacterial
resistance is the major cause of failure of antibiotic
therapy requiring additional courses of protracted
antibiotic usage and in effect setting up a vicious cycle
(Hopkins et al., 2005).
In the present study both Gram negative rods {S.
typhimurium (ATCC 14028), MDR S. typhi} were more
sensitive to AGE than S. aureus (ATCC 25923). This
demonstrates that Gram positive organisms may be
Hannan et al. 4477
better protected naturally against the action of garlic
extract because of the presence of thick layer of
peptidoglycan which impairs the access the antibacterial
agent (allicin) (Bakri and Douglas, 2005; Indu et al.,
2006).
A comparable study done on AGE, also demonstrated
the zone of inhibition of 23.8±1.5 mm against 10 MDR S.
typhi isolates (Iwalokun et al., 2004). In contrast, a
Nigerian study demonstrated different results which
showed that no zone of inhibition was formed by aqueous
extract against S. typhi (Ekwenye and Elegalam, 2005).
This variation may be due to the fact that garlic
composition depends upon many factors like origin, age,
storage conditions and method used for the preparation
of extract which may in turn its activity (Sivam, 2001).
In case of MIC, more MDR isolates were inhibited at 22
mg/ml than sensitive isolates, illustrating that some type
of resistant may also exists in MDR isolates. But in
contrast to this observation both MDR (n=30) and
sensitive (n=20) isolates were inhibited within same
range that is 18-22 mg/ml. It might be due to the
difference in mechanism of action of garlic; antibiotics
have a single mode of action and it is 1000-fold easier to
develop resistance against antibiotics drugs. On the other
hand garlic has multiple mechanisms due to its various
constituents that give their effects simultaneously (Jabar
and Al-Mossawi, 2007; Bakri and Douglas, 2005). This
showed that garlic was equally effective against MDR
and sensitive isolates.
In the present study, the MIC range for 50 S. typhi
isolates came out to be 18 - 22 mg/ml, by agar dilution
method. In Nigerian study, MIC range for 10 MDR S.
typhi isolates was 16-27 mg/ml (Iwalokun et al., 2004).
However, another study proved that S. typhi isolate was
not inhibited at even 1000 mg/ml (Ekwenye and
Elegalam, 2005). The most probable explanation for
these differences are the amount and composition of
organo-sulfur compounds which vary with different
species of garlic (Arora and Kaur, 1999).
The basic ground for selecting AGE for this study was
because of high concentration of allicin (antibacterial
component) as compared to other extracts (Jabar and Al-
Mossawi, 2007). AGE could be conveniently utilized in-
vivo studies (Kemper, 2000). The major drawbacks in its
use are that the amount of allicin decreases with time.
Thus it cannot be preserved for longer time and its
antibacterial properties also decline with the age of
extract. Therefore, fresh AGE should be prepared each
time before its use (Pranoto et al., 2005).
One of the disadvantages in evaluating antibacterial
activity of AGE is lack of standardization in techniques
being used by the scientists. This gives rise to marked
difference in results obtained.
The most likely rationalization for this difference is may
be due to existence of variation in the component of
different origin. A study by Lawson et al., (1991)
established that the garlic cropped in China may have
4478 Afr. J. Microbiol. Res.
Table 2. Anti-bacterial activity of AGE in agar well diffusion assay (Zone of inhibition).
Dilutions of AGE (mg/ml)
Zones of inhibition (mm)
MDR S. typhi isolates
S. aureus ATCC (25923)
S. typhimurium ATCC (14028)
Stock solution (500)
34±1
38±1.5
36±2
1/2(250)
26±1
32±1
31±1.5
1/4(125)
21.5±1.5
25±1
29±1.5
1/8(62.5)
16.5±1
-
25±1
1/16(31.25)
12.5±1.5
-
16±1
*AGE; Aqueous garlic extract, MDR; multi-drug resistant; ATCC; American type culture collection.
Table 3. Inhibition of MDR and non-MDR S. typhi isolates with AGE (mg/ml).
AGE (mg/ml)
Sensitive isolates (n=20)
MDR isolates (n=30)
18
09
04
19
04
05
20
04
06
21
02
06
22
01
09
Table 4. MIC of AGE against MDR (n=30) and sensitive (n=20) S. typhi isolates.
S. typhi isolates
MIC range (mg/ml)
MIC50 (mg/ml)
MIC90 (mg/ml)
MIC100 (mg/ml)
MDR isolates
18-22
20
>21
22
Sensitive isolates
18-22
>18
>20
22
Figure 1. Anti-bacterial activity of garlic showing zone of inhibition on plate.
Hannan et al. 4479
Figure 2. Cumulative percentage of multi-drug resistant S. typhi (n=30) inhibited at different concentrations of
garlic extract.
twice allicin as much as in Europe or United States.
Another reason which also effected upon results is type
of methodology adopted for the assessment of screening
and MIC of the extract. A number of factors namely,
depth of agar, pH, lag-time required for the diffusion of
extract, size of inoculum and length of incubation may
influence the MIC of the extract (Pranoto et al., 2005).
The shortcomings in different techniques demand
standardization so that inter-study outcome can be safely
measured up. Lastly it is important to develop guidelines
for all procedure adopted in evaluating antibacterial
activity of AGE. It is also a need of hour to investigate
extracts of allium species of different geographical
locations for the most active ingredients responsible for
their antibacterial activity.
Conclusion
Aqueous garlic extract was found to be inhibitory against
the isolates of S. typhi. It is worth describing that garlic
might be utilized as anti-typhoid agent after determining
its pharmacokinetics and pharmacodynamics.
ACKNOWLEDGEMENT
We are grateful to University of Health Sciences, Lahore
Pakistan for financial and logistic support for this
research project.
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