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Research Article
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Synergic Interactions Between Selected Botanical Extracts and Tetracycline Against Gram Positive and Gram Negative Bacteria
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W.A. Hussin
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W.M. El-Sayed
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ABSTRACT
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The potential use of plants as antimicrobial agents in medicine has not been fully exploited. The present study was carried out to investigate the synergic interaction of dichloromethane/methanol extracts from Punica granatum (PG), Thymus vulgaris (TV), Commiphora molmol (CM) and Achillea fragrantissima (AF) with tetracycline against Gram positive (Staphylococcus aureus ATCC 25923 and Bacillus megaterium ATCC 14591) and Gram negative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Klebsiella pneumonia ATCC 700603) bacterial strains. All extracts had a significant antibacterial activity against all tested bacteria within concentrations range of (0.1-50 mg/disk). The lowest Minimum Inhibitory Concentration (MIC) recorded was 1 mg mL-1. The recorded MICs values of PG and TV against Gram negative bacteria were higher than those against Gram positive strains. Combination of PG, TV, or CM with tetracycline achieved synergetic interaction against tested bacteria as indicated from fractional inhibitory concentration and confirmed by time kill assays. CM, PG and TV significantly increased the Post Antibiotic Effect (PAE) of tetracycline from 1 h to 3, 4 and 5 h, respectively. The results suggested the use of these plants in extending the lifetime of fading antibiotics.
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Received: November 15, 2011;
Accepted: November 23, 2011;
Published: December 10, 2011
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INTRODUCTION
The failure of antibiotics therapy for many pathogens has revived the search
for antimicrobial compounds from natural sources. Plant extracts have played
an important role in the potential discovery of new antimicrobial agents for
treatment of infectious diseases (Darwish et al.,
2002; Konning et al., 2004; Karim
et al., 2011; Sohail et al., 2011).
Hundreds of plants have been used worldwide in traditional medicine for controlling
bacterial infections (Martin and Ernst, 2003). Several
studies have proposed that natural compounds in combination with antibiotics
could offer a new strategy for developing therapies against bacterial infections
through potentiating the activity of antibiotics (Braga et
al., 2005).
Pomegranate (Punica granatum), myrrh (Commiphora molmo), thyme
(Thymus vulgaris) and gaysum (Achillea sp.) are used in folk medicine
for decades. Fruits, peels and roots of pomegranate have been commonly used
in herbal remedies in many countries. The antimicrobial activity of peels has
been demonstrated against pathogenic bacteria including S. aureus, Salmonella
paratyphi, Shigella dysenteriae, E. coli, P. aeroginosa,
K. pneumonia, Streptococcus pneumonia and Listeria monocytogenes
and Bacillus subtilis (Braga et al., 2005;
Reddy et al., 2007; Al-Zoreky,
2009; Hayouni et al., 2011). In addition,
P. granatum was effective at inhibiting the growth of multiresistant
strains of S. aureus which acquired resistance to all conventional antibiotics
among hospitalized patients (Machado et al., 2003;
Melendez and Capriles, 2006). C. molmol is used
as an antiseptic and anti-inflammatory for the topical treatment of mouth and
throat infections (Kumari et al., 2011). Different
extracts of Commiphora sp. showed antimicrobial potency against Gram
negative and Gram positive bacteria (Suleiman et al.,
2010). Also, the essential oils of the plant exerted antimicrobial activity
especially against Gram positive bacteria (Mothana et
al., 2010). T. vulgaris is a well known medicinal plant frequently
used as a tea or as an additive in commercial spice mixtures of many foods for
aroma and flavor (Sokovic et al., 2010). There
are numerous reports on antibacterial activities of the essential oils from
various Thymus species against human and food pathogenic bacteria (Couladis
et al., 2004; Kotan et al., 2010).
The antibacterial activities of the essential oils of some Achillea species
against various bacteria have been previously reported (Kotan
et al., 2010). In addition, the methanolic extract of some species
of Achillea plant enhanced the inhibitory effects of different antibiotics
including, chloramphenicol, neomycin, doxycycline, cephalexin and nalidixic
acid against the growth of sensitive and resistant strains of E. coli
(Darwish and Aburjai, 2010).
The present study aimed to evaluate the antimicrobial effect of dichloromethane
/ methanol extract of Punica granatum, Thymus vulgaris, Commiphora
molmol and Achillea fragrantissima against Gram positive and Gram
negative bacteria. The Minimum Inhibitory Concentration (MIC) of each plant
extract was determined. To examine the potentiating activity of these botanical
extracts on the effect of antibiotic to combat bacterial infections, synergistic
interaction between the examined plant extracts and tetracycline was carried
out and the Fractional Inhibitory Concentration (FIC) was calculated. The results
of synergistic interaction assays were confirmed by growth kill assays. The
effect of plant extracts on the Post Antibiotic Effect (PAE) of tetracycline
was assessed. Post Antibiotic Effect (PAE) is the term used to describe suppression
of bacterial growth that persists after brief exposure of organisms to antimicrobials.
The increased PAE could offer an alternative way for the extension of the useful
lifetime of antibiotics (Spangler et al., 1998).
MATERIALS AND METHODS Chemicals: All reagents were purchased from Sigma (St. Louis, MO) except where indicated in the specified methods. Plant materials: The oleo-resin of C. molmol, leaves of T. vulgaris, barks of A. fragrantissima and peel of P. granatum were purchased from a local market (Alhasa, KSA) and identified with the help of a specialized taxonomist. Extraction of the plant material: Extraction of the plants was performed using continuous technique in which the specific parts of the plants were dried and milled. The powder of each plant was extracted with dichloromethane: methanol (1:1 v/v) at room temperature for 3 days and filtered. The solvent was then evaporated under vacuum. Bacterial strains: The antibacterial assay was performed against standard strains of Gram positive (S. aureus ATCC 25923 and B. megaterium ATCC 14591) and Gram negative (E. coli ATCC 25922, P. aeruginosa ATCC 27853 and K. pneumoniae ATCC 700603) bacterial strains. All strains were purchased from ATCC (Manassas, VA). The strains were cultured aerobically overnight on nutrient agar (Oxoid, UK) at 37°C and then frozen at -80°C in 30% glycerol solution (Pro-labs, UK) until used. In all assays, inocula were prepared using overnight cultures on nutrient agar that were then suspended in nutrient broth to a turbidity equivalent to 0.5 McFarland containing 1.5x108 cell forming unit mL-1 (CFU mL-1). Antimicrobial activity of plant extracts by disk diffusion method Overnight growing cultures were adjusted to match 0.5 McFarland standard and then diluted 1:10 using Mueller-Hinton broth (Difco Laboratories, Detroit, MI) and used for inoculation of agar media. The seeded agar media were immediately mixed and poured in Petri plates and allowed to solidify. Filter paper disks (Whatman No.1, 6 mm in diameter) were loaded with different concentrations (0.1, 0.5, 1, 10, 50 mg/disk) of different plant extracts. The loaded disks were placed over agar surface and left in refrigerator for 2-3 h to allow diffusion of the extracts. All plates were inverted and incubated at 37 °C for 24 h. Following incubation, the mean diameter of inhibition zones around the disks was measured to the nearest mm. All experiments were carried out in triplicates.
Minimum Inhibitory Concentration (MIC) of the tested botanical extracts:
The minimum inhibitory concentration of plant extracts was determined by broth
microdilution method according to National Committee for Clinical
Laboratory Standards (1999). All strains were cultured on Muller Hinton
agar (Oxoid, UK) and incubated at 37°C for 24 h prior to MIC determination.
An inoculum density of 105 CFU mL-1 of each of the test
organisms was prepared in Muller Hinton Broth (MHB). MHB was dispensed into
wells of a 96- well microtitre plate. A stock solution of each plant extract
was prepared and diluted in DMSO. A DMSO control was included in all assays.
Plant extracts were serially diluted in the wells followed by the addition of
the bacterial inocula. Control samples were performed simultaneously by growing
different bacterial strains without the plant extracts. The microtitre plates
were incubated at 37°C for 18 h. The plates were subjected to reading at
600 nm using plate reader. The MIC value was recorded as the lowest concentration
at which no growth was observed. All MIC values were determined on three independent
experiments.
Synergic interaction between plant extracts and tetracycline against tested bacterial strains: To estimate the effect of the tested botanical extracts in combination with antibiotics on the growth of used bacterial strains, increasing concentrations of tetracycline (0.5 to 128 μg mL-1) were prepared. The prepared concentrations of tetracycline were added to MHB inoculated with 105 CFU mL-1 of each test strain. The botanical extracts were prepared at 0.25xMIC and added to each plate. The assay was carried out for tetracycline alone and each plant extract as control. The plates were incubated at 37°C for 24 h, the lowest antibiotic concentration in combination with plant extract that prevented the development of turbidity was regarded as the MIC*.
Determination of the fractional inhibitory concentration (FIC): The
activity of antibiotic and each plant extract in combination was determined
by calculating the Fractional Inhibitory Concentration (FIC) index as described
by Mackay et al. (2000) as follows: FIC of drug
A = (MIC* drug A+Plant extract) / MIC drug A alone, FIC ≤0.5 indicating synergy,
FIC>0.5 to 4 means indifference and FIC>4 indicating antagonism.
Effect of selected botanical extracts and tetracycline on the growth of Escherichia coli ATCC 25922: Growth curves for E. coli ATCC 25922 were performed in MHB in the presence of either plant extract (0.25xMIC), tetracycline (0.5xMIC), or both. A standard inoculum of approximately 105 CFU mL-1 was used and then the tubes were incubated at 37°C on a shaking-platform incubator for 24 h. At various time intervals (0, 4, 8, 12, 16 and 24 h) during the incubation period, samples were subjected to a 10-fold serial dilutions and 0.1 mL aliquots of these dilutions were spread onto nutrient agar and incubated at 37°C for 24 h for viable cell count or CFU determination.
Post antibiotic effect (PAE): E. coli ATCC 25922 strain was grown
overnight at 37°C and then diluted with the MHB and further incubated at
37°C for 2-3 h to enter the logarithmic growth phase. The actively growing
culture was subsequently diluted with MHB to achieve turbidity matching that
of a 0.5 McFarland standard. The inocula equivalent to 104 CFU mL-1
were incubated at 37°C for 30 min with or without tetracycline (1xMIC),
the plant extract alone (0.25xMIC), or a combination of antibiotic and the plant
extract. After 30 min, bacteria were diluted 1000 fold with MHB to eradicate
the antibiotic and/or plant extracts. Control experiments were performed simultaneously
for cells unexposed to either antibiotic or plant extracts. A 100 μL of
each treatment was obtained at time 0 and every hour thereafter until turbidity
developed and spread onto nutrient agar medium for total viable cell count determination.
The number of CFU mL-1 was determined after incubation at 37°C
for 24 h. PAE was calculated as described by Craig and Gudmundsson
(1996): PAE = T-C, where T is the time required for the viable cell counts
of the exposed bacteria to increase by a factor of log10 from the
initial time immediately after washing and C is the corresponding time for cells
unexposed to either antibiotic or plant extract. Each assay was carried out
in triplicates and results were expressed as the means of PAE values. To avoid
the overgrowth of control culture, a 100-fold dilution was prepared using MHB
at the same time points as scheduled for antibiotic exposures during the study.
RESULTS Antimicrobial activity of plant extracts by disk diffusion method: In the present study, the antimicrobial activity of dichloromethane/methanol extracts from PG, TV, CM and AF was tested against Gram positive and Gram negative bacterial strains (Table 1). The results showed that PG extract exhibited a potent antibacterial activity against all tested strains within the used range of concentrations (0.5-50 mg/disk), producing 10-32 mm inhibition zone in a dose-dependent manner. TV extract inhibited the growth of E. coli ATCC 25922 and S. aureus ATCC 25923 at concentrations of 10 and 50 mg/disk. In addition, it also inhibited the growth of B. megaterium ATCC 14591 and P. aeruginosa ATCC 27853 at concentrations ranging from 1-50 mg/disk but had no inhibitory effect on the growth of K. pneumonia ATCC 700603 at any of the concentrations investigated. CM and AF extracts had no effect on E. coli ATCC 25922 and were effective against S. aureus ATCC 25923 at 10-50 and 50 mg/disk, respectively. Similarly, the inhibition in the growth of B. megaterium ATCC 14591 was achieved at concentrations of 10-50 and 50 mg/disk of CM and AF, respectively. CM had better efficiency against the growth of P. aeruginosa ATCC 27853 and K. pneumonia ATCC 700603 at low extract concentrations (0.5-50 mg/disk). AF was unique among all extracts under investigation in inhibiting the growth of P. aeruginosa ATCC 27853 at 0.1 mg/disk. It also inhibited the growth of K. pneumonia ATCC 700603 at the highest two concentrations studied; 10 and 50 mg/disk (Table 1). AF was also unique being the extract with the least antimicrobial potency.
Minimum Inhibitory Concentration (MIC): All botanical extracts were
assessed for MIC determination using broth microdilution method using Muller
Hinton broth medium. The MIC values of the tested extracts varied with the bacterial
strain tested, ranging from 1- 256 mg mL-1 (Table 2).
Table 1: |
Antimicrobial activity of tested plant extracts by disk-diffusion
method. |
 |
*PG: Punica granatum, TV: Thymus vulgaris, CM:
Commiphora molmol, AF: Achillea fragrantissima ; no inhibition.
The data are expressed as the means of three independent experiments |
Table 2: |
Minimum inhibitory concentration (MIC) of the tested botanical
extracts against different bacterial strains |
 |
+The data are expressed as the means of three independent
experiments |
PG was most effective against B. megaterium with MIC value of 1mg mL-1
and least effective against E. coli with MIC value of 64 mg mL-1.
The recorded MICs of PG against the growth of S. aureus, K. pneumonia
and P. aeruginosa were 8, 16 and 32, respectively. CM was most effective
against K. pneumonia and S. aureus with MIC values of 1 and 8
mg mL-1, respectively. E. coli, P. aeruginosa and
B. megaterium required higher concentration of CM to be inhibited and the
recorded MIC was 16 mg mL-1. TV exerted higher inhibitory activity
against Gram positive bacteria with MIC values of 4 and 16 mg mL-1
against the growth of B. megaterium and S. aureus, respectively.
The reported MIC values of TV for K. pneumonia, E. coli and
P. aeruginosa were 16, 32 and 32, respectively.
Table 3: |
Synergic interaction between the tested botanical extracts
and tetracycline against the tested bacterial strains |
 |
+PG: Punica granatum, TV: Thymus vulgaris,
CM: Commiphora molmol MIC: Minimum inhibitory concentration of
tetracycline. MIC*: Minimum inhibitory concentration of tetracycline (0.5
X MIC) in combination of plant extract (0.25xMIC). FIC: Fractional inhibitory
concentration (MIC*/ MIC of tetracycline). FIC≤0.5 indicating synergy,
FIC>0.5 to 4 means indifference and FIC > 4 indicating antagonism.
The data are expressed as the means of three independent experiments |
AF extract showed the least antibacterial activity with high MICs ranging
from 64 to 256 mg mL-1 against the bacterial strains under investigation
(Table 2). Overall, CM was the most effective extract with
low MIC values against most bacterial strains.
Synergic interaction between botanical extracts and tetracycline: The
interaction of PG, TV, or CM with tetracycline was investigated against the
growth of the bacterial strains under investigation (Table 3).
The reported MIC values for tetracycline were 4 μg mL-1 for
B. megaterium, 8 μg mL-1 for S. aureus, E. coli and
P. aeruginosa and 16 μg mL-1 for K. pneumonia. The
effect of combination of each plant extract at a concentration of 0.25xMIC with
0.5xMIC of tetracycline was examined. The lowest antibiotic concentration in
combination with the plant extract that prevented the development of turbidity
was regarded as the MIC* and the Fractional Inhibitory Concentration (FIC) index
was calculated. Indifference (FIC = 4) was shown with PG extract against
P. aeruginosa ATCC 27853. Combination of PG extract with tetracycline achieved
synergistic activity against the other tested strains with FIC ranging from
0.063 to 0.5. Co-treatment of all tested bacterial strains with TV and tetracycline
increased the antibacterial activity of tetracycline as indicated from FIC values
(0.031-0.5). CM had no effect on the activity of tetracycline against E.
coli (FIC = 1). However, it showed a significant synergy in inhibiting the
growth of all other bacterial strains tested with FIC 0.063-0.25 (Table
3).
Effect of selected plant extracts and tetracycline on the growth of Escherichia coli ATCC 25922: For time-kill assays, growth curves for E. coli ATCC 25922 were performed in the presence of either plant extract (0.25xMIC), tetracycline (0.5xMIC), or both. The total viable cell count in relation to time was determined. Treatment of bacteria with either tetracycline, PG or TV were similar to the control in starting the exponential phase of growth after 4 h, while the combination of PG or TV and tetracycline extended the lag phase and prevented the growth for the following 24 h (Fig. 1 and 2). Using CM extract with tetracycline (Fig. 3) had no significant effect on tetracycline activity which in agreement with the synergistic study.
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Fig. 1: |
Growth curves of Escherichia coli ATCC 25922 (control),
in the presence of Punica granatum (PG) (0.25 MIC), tetracycline
(0.5 MIC), or a combination of the plant extract (0.25 MIC) and tetracycline
(0.5 MIC). Data are expressed as the mean of three independent assays |
Post Antibiotic Effect (PAE): The post antibiotic effect induced by
the investigated botanical extracts was carried out using E. coli ATCC
25922. The tested bacteria was exposed to tetracycline (1xMIC), each of the
plant extract (0.25xMIC) and a combination of each plant extract with tetracycline
for 30 min. Control experiments were performed simultaneously for cells unexposed
to tetracycline or plant extracts. The change in total viable cell count over
time under the exposure conditions was represented in (Fig. 4-6).
The estimated PAEs for tetracycline, PG, TV, or CM were 1, 1, 3 and 0 h, respectively.
The combination of tetracycline and PG, or TV resulted in a 4-fold increase
in PAE (from 1 to 4 h) for PG and a 5-fold increase in PAE (from 1 to 5 h) for
TV (Fig. 4 and 5).
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Fig. 2: |
Growth curves of Escherichia coli ATCC 25922 (control),
in the presence of Thymus vulgaris (TV) (0.25 MIC), tetracycline
(0.5 MIC), or a combination of the plant extract (0.25 MIC) and tetracycline
(0.5 MIC). Data are expressed as the mean of three independent assays |
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Fig. 3: |
Growth curves of Escherichia coli ATCC 25922 (control),
in the presence of Commiphora molmol (CM) (0.25 MIC), tetracycline
(0.5 MIC), or a combination of the plant extract (0.25 MIC)and tetracycline
(0.5 MIC). Data are expressed as the mean of three independent assays |
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Fig. 4: |
The post-antibiotic effect (PAE) of Punica granatum
(PG) (0.25 MIC), tetracycline (1 MIC), or a combination of the plant extract
and tetracycline on Escherichia coli ATCC 25922. Control curve is
representing the growth of bacteria unexposed to either the plant extract
or tetracycline. Data are expressed as the mean of three independent assays |
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Fig. 5: |
The post-antibiotic effect (PAE) of Thymus vulgaris
(TV) (0.25 MIC), tetracycline (1MIC), or a combination of the plant extract
and tetracycline on Escherichia coli ATCC 25922. Control curve is
representing the growth of bacteria unexposed to either the plant extract
or tetracycline. Data are expressed as the mean of three independent assays |
Although CM alone was not as effective as the other two plant extracts and
showed no difference in PAE as compared to unexposed cells, the combination
of CM extract with tetracycline resulted in a 3-fold increase in the PAE (Fig.
6).
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Fig. 6: |
The post-antibiotic effect (PAE) of Commiphora molmol (CM)
(0.25 MIC), tetracycline (1MIC), or a combination of the plant extract and
tetracycline on Escherichia coli ATCC 25922. Control curve is representing
the growth of bacteria unexposed to either the plant extract or tetracycline.
Data are expressed as the mean of three independent assays |
DISCUSSION
The antimicrobial activities of dichloromethane/ methanol extracts of PG, TV,
CM and AF were evaluated against Gram positive and Gram negative bacteria. The
inhibitory activity of the tested extracts was initially determined by disk-diffusion
method. Marked microbial inhibition was recorded with PG against all tested
bacterial strains (Table 1) reflecting a broad spectrum activity
of the plant extracts which in accordance with previous reports (Prashanth
et al., 2001; Al-Zoreky, 2009). All tested
strains were susceptible to TV extract except K. pneumonia. CM and AF
were effective in inhibiting the growth of Gram positive bacteria (B. megaterium
and S. aureus) but only at high concentrations 10 and 50 mg/disk. Also,
CM and AF showed no activity against Escherichia coli (Table
1). In general E. coli was the least affected strain while Pseudomonas
aeruginosa was affected by all extracts at most concentrations investigated.
The antibacterial activity of CM may be attributed to the high terpenes content
of the oleo-resin of the plant (Rahman et al., 2008).
The lowest concentration of the tested plant extract that prevented the growth
of the tested bacteria was investigated using broth microdilution method and
defined as MIC. The recorded MIC values of PG and TV against Gram negative bacteria
(Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumonia)
were higher than those for Gram positive strains (Staphylococcus aureus
and Bacillus megaterium) reflecting that tested Gram positive bacteria
were more sensitive than Gram negative bacteria to both botanical extracts.
This is in agreement with Holetz et al. (2002)
who reported that PG showed significant activity against S. aureus and
no activity against E. coli and P. aeroginosa. CM was the most
effective botanical extract with high potency against the growth of all bacterial
strains. The lowest MIC (1 mg mL-1) was recorded with PG and CM extracts
against B. megaterium ATCC 14591 and K. pneumonia, respectively
(Table 2) while the highest MIC (64- 256 mg mL-1)
was recorded for AF with all tested bacterial strains. The low activity of different
extracts from Achillea sp. was reported against K. pneumonia, P.
aeruginosa, S. aureus and E. coli (Tuberoso
et al., 2005; Konakchiev et al., 2011).
The high values of MIC recorded with AF against the tested bacteria indicating
low activity, suggested the elimination of this extract from further investigations.
The interaction between the tested botanical extracts and tetracycline against
the tested bacterial strains were evaluated. Tetracycline has been used since
1940s as a broad-spectrum antibiotic in the treatment of a wide variety of bacterial
infections including the infections of respiratory tract, sinuses, middle ear,
urinary tract and intestines. It is also used in the treatment of gonorrhea,
especially in patients allergic to β-lactams and macrolides (Cunha
and Garabedian-Ruffalo, 1990). The general usefulness of this antibiotic
has been reduced with the onset of bacterial resistance (Roberts,
2005). To investigate the effect of the botanical extracts on the potency
of tetracycline in inhibiting the bacterial growth, the interaction between
tetracycline and the botanical extracts was evaluated. Initially, the MIC values
of tetracycline were determined against the tested bacteria. The susceptibility
testing was performed using growth microdilution method. In the combination
assays, the used concentration of each plant extract was 0.25xMIC since higher
concentration significantly reduced the total viable count of the tested bacteria
(data not shown). In a similar way, the used concentration of tetracycline was
0.5xMIC. The interaction between tetracycline and the investigated botanical
extracts was determined by calculating the fractional inhibitory concentration
(Table 3). Synergy activity was recorded between PG extract
and tetracycline against the tested bacteria (FIC: 0.063 to 0.5) except for
P. aeruginosa ATCC 27853 with FIC = 4. PG extract had a significant effect
on improving the activity of tetracycline which was previously reported by Braga
et al. (2005). Treatment of bacteria with TV and tetracycline increased
the antibacterial activity of tetracycline as indicated from FIC values (FIC
0.031- 0.5). Fujita et al. (2005) demonstrated
that crude extract of Thymus leaves significantly increased the activity
of tetracycline against MRSA clinical isolates. CM had no effect on the activity
of tetracycline against E. coli (FIC = 1). However, it showed a significant
synergy in inhibiting the growth of all other bacterial strains (FIC: 0.063-0.25).
The displayed potentiation exerted by CM extract on tetracycline activity against
S. aureus and K. pneumonia was reported by Rahman
et al. (2008).
To confirm the possible use of synergistic antibiotic-herb combination for
combating infections caused by a Gram negative pathogen , time kill assays for
Escherichia coli ATCC 25922 in the presence of either plant extract (0.25xMIC),
tetracycline (0.5xMIC), or both were performed. Escherichia coli was
selected as one of the most causes of urinary tract infections (Bosch
et al., 2011). Based on our findings, Escherichia coli was
the least susceptible organism to the investigated plant extracts with high
MIC values. Time kill assays were carried out to confirm the interaction between
the botanical extracts and tetracycline. Treatment of bacteria with either tetracycline,
PG or TV were similar to the control in starting the exponential phase of growth
after 4 h, while the combination of PG or TV and tetracycline extended the lag
phase and prevented the growth for the following 24 h (Fig. 1
and 2). Using CM extract with tetracycline (Fig.
3) had no significant effect on tetracycline activity which in agreement
with synergistic study. Very few reports concerning the effect of medicinal
plants in general and especially the plants under investigation concerning their
effect on the synergism of antibiotics.
To date, many studies have been concerned with bactericidal or bacteriostatic
activity of different plant extracts. However, it is important to consider the
sustainability of tested antimicrobial extracts over a long period of time.
The Post Antibiotic Effect (PAE) is the term used to describe the suppression
of bacterial growth that persists after brief exposure of microorganism to antimicrobial
agents. Drugs with no PAEs may require more frequent administration than those
with PAEs (Spangler et al., 1998). The PAE of
tetracycline, each of plant extract, or combination of both was tested. The
estimated PAEs for tetracycline, PG, TV, or CM were 1, 1, 3 and 0 h, respectively.
PG was as effective as tetracycline in delaying the growth of E. coli,
while TV was more effective than the antibiotic resulting in a 3-fold increase
in time required for E. coli to grow by log10. The combination
of tetracycline and PG, or TV extended the PAE to 4 and 5 h, respectively. CM
alone was not as effective as PG or TV extracts and caused no difference in
PAE as compared to unexposed cells. However, the combination of CM extract with
tetracycline resulted in a 3- fold increase in the PAE. The increased PAE could
help reducing the dosing regimens time and cost treatment and represent an alternative
way for the extension of the useful lifetime of antibiotics. The availability
of literature in this specific area was challenging, the search for post antibiotic
effect and plant extracts on Pubmed.gov yielded only 9 abstracts. The broad
spectrum antibacterial activity of the investigated botanical extracts and their
role in potentiating antibiotic activity suggested the use of these plants in
combating bacterial infections and in extending the lifetime of some antibiotics
that lost efficiency or have fading antibacterial activity.
ACKNOWLEDGMENT This project was supported by King Faisal University, Deanship of Scientific Research, Grant 10084. The authors would like to thank Prof. Sayed Gogheiman for helping identifying the plants.
|
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