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Susceptibility
of Drug of
Resistant
Acinetobacter
baumanii
(DRAB)
totoa astabilized
Susceptibility
Drug Resistant
Acinetobacter
baumanii
(DRAB)
stabilized
aqueous
allicin
extract
from
garlic
(AB1000
).
aqueous
allicin
extract
from
garlic
(AB1000
).
Cutler RR , Wilson P , Thota MR , Vivekananthn S , Bennett NJ , and Josling, PD
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2
1
1
3
3
Researchers’/Presenters’
Names
1School of Health and Bioscience, University of East London, UK, 2Dept of Medical Microbiology, The Royal London and St Bartholomew’s Hospitals, London, UK, 3 Allicin International, Rye, UK
Institution/Organization/Company
Figure 1: HPLC of allicin extract
Abstract
Methods
1.00E+08
Methods: The antimicrobial activity of a novel aqueous allicin extract (AB1000) was tested against 11
clinical isolates of DRAB. The allicin content of AB1000 was confirmed using HPLC. Strains were screened
for activity using agar diffusion methods, MICs and MBCs were carried out and growth (using
spectroscopy at 490nm) and killing curves (using viable counts) for selected organisms were determined.
Results: In agar diffusion tests using AB1000 concentrations ranging from 125 to 1000mg/l were made up
in aqueous solution. Zone diameters on Oxoid Muller Hinton agar ranged from 14mm to 31mm, slightly but
not significantly smaller than the zones found with Oxoid Isosensitest agar of 20mm to 34mm. At 500mg/l
the zone sizes for the 11 strains tested varied from 22-29mm with an average zone diameter of 26.3 mm
and a mode diameter of 27mm. Minimum inhibitory concentrations varied from 15 mg/l to 62.5mg l -1 with a
mode concentration of 62.5 mg l-1. Minimum bactericidal concentrations varied from 62.5 to 125 mg l-1. In
growth curves, when compared to the allicin free control, growth at sub-inhibitory concentrations was
delayed by 1-2hrs at 15 mg l-1 and by 3-4hrs at 31mg l-1. At 62.5 mg l-1 growth was completely inhibited.
In killing curves using AB1000 at a concentration of 500mg l-1, growth was reduced by 30% in 1 hour and
by 99.9997% at six hours. This represented a reduction in cfu ml-1 from Log 7.3 to Log 3.8 cfu ml-1. At
24hrs no growth was detected.
1.00E+07
1.00E+06
cfu/ml
The antimicrobial activity of a novel aqueous allicin extract (AB1000) was tested
against 11 isolates of DRAB isolated from patients at the Royal London Hospital.
Allicin was extracted using a patented cold aqueous extraction method. The allicin
content of AB1000 was confirmed using HPLC (Fig 1). Strains were screened for
activity using agar diffusion methods on 3 media, isosensitest, nutrient and Mullerhinton (all Oxoid Ltd,UK). Individual plates were lawned with each test organism using
the BSAC standard method, 6mm holes were cut in the centre of the plate, 100ul of
each dilution of allicin liquid was added to the hole and plates incubated at 370C
overnight. Inhibition zones sizes were measured the next day. Growth curves, MICs
and MBCs were carried out. Growth was determined using spectroscopy (at 490nm)
and killing curves were determined (using viable counts and 500mg ml-1 allicin).
Objectives: DRAB is a nosocomial pathogen. Globally many Intensive Care Units have their own endemic
strain. Some patients become colonised with the organism, with no adverse effect, whereas others have
life-threatening infections. DRAB can be resistant to many antimicrobial agents and treatment of infections
is increasingly difficult due to the dwindling choice of active agents. There is an urgent need for new
agents active against DRAB and our objective was to contribute to this search.
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
(162)
1.00E+00
0
Results
Introduction
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2.5
2.5
Abs
LH3
1.5
1
0.5
0
0
2
0
Time(hrs)
5
0
10
25
LH3
LH4
LH6
We have shown that our aqueous allicin extract is active in vitro against a range of
clinical isolates of DRAB. Allicin can inhibit the growth of DRAB at concentrations as
little as 31 to 62mg l-1. We have also shown Allicin can be bactericidal against DRAB
1.5
0
15
20
Allicin has previously be shown to be active against multiply drug resistant organisms.
Extracts of garlic have also been demonstrated to act as quorum sensing inhibitors
reducing the formation of bacterial biofilms.
0.5
10
15
Conclusions
1
3
2
1
50
Abs
Abs
2
10
Figure 1 shows demonstrates the purity of the extract over 98% pure allicin. Figure2
shows that in liquid culture, grow is inhibited at 62.5mg l-1. but below that some strains
grow more slowly at 31 or 15 mg l-1. Using 500 15 mg l-1 of allicin (Fig 3) DRAB
showed reduced growth (30%) after 1hr. There was no growth at 24hrs.
Agar Dilution Tests : Zone sizes were greatest using isosensitest agar and least using Nutrient agar. At 500mg l-1
(ppm) the average zone size for all 11 strains was 26.3mm
(+/- 3mm)on iso-sensitest agar.
3
5
hours
LH2
Conclusion: Allicin (AB1000) was shown to be bactericidal against DRAB at pharmacokinetically
achievable concentrations. Killing curve data shows antibacterial activity begins within the first hour of
contact. We have also demonstrated that sub-inhibitory concentrations as low as 15mg l-1 can reduce
growth.
Acinetobacter an important nosocomial pathogen. It can grow at various temperatures and
pHs, and carries resistance to many antibiotics. Acinetobacter is common in the
environment, in soil and water and in addition found on the skin and distal urethra of
healthy people. It is also part of the normal skin flora. Acinetobacter has a very high level
of resistance to antimicrobials, and relatively few antibiotics are active against it. There is a
strain of highly resistant Acinetobacter that is causing wound infections in injured troops in
Iraq 1. The respiratory tract is the most common site of infection. Acinetobacter can cause
a nosocomial pneumonia, which is often associated with ventilator use. Other predisposing
factors for Acinetobacter pneumonia include intensive care residence, intubation,
tracheostomy, antibiotic therapy, surgery, and underlying pulmonary disease. Allicin is the
main biologically active agent produced in garlic. It is normally very unstable however this
aqueous extract is stable for up to 2yrs as a powder . We have previously demonstrated
that it can be active against multiply drug resistant bacteria such as MRSA 2, 3 and this
study shows its activity against DRAB
Figure 3: Comparative Killing curves
against 4 strains of DRAB
5
15
10
Time(hrs)
Time(hrs)
1000 ppm
500 ppm
250 ppm
125 ppm
62.5 ppm
31.25 ppm
15.6 ppm
7.8 ppm
3.9 ppm
1.95 ppm
0.97 ppm
Control
Figure 2: Growth curves for LH3 (left) and LH6 (right) showing the effects
of different concentrations of allicin
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Further work is being carried out to determine if allicin inhibits the formation of biofilms
by DRAB and how allicin can work in combination with other antibiotics against this
multiply – drug resistant organism.
References
1. Davis KA, Moran KA, McAllister CK, Gray PJ. (2005) Multidrug-resistant Acinetobacter extremity infections
in soldiers. Emerg Infect Dis. 11(8):1218-24
2. Cutler, RR., Wilson,P. (2004) Antibacterial activity of a new, stable, aqueous extract of allicin against methicillinresistant Staphylococcus aureus. Brit.J.Biomed.Sci.:61: 71-74
3. Cutler RR, Josling PD and Bennett NJ (2005) Treatment of chronic MRSA infections using a novel aqueous extract of
Allicin (AB1000). Clinical Microbiology and Infection. 11: suppl2. p515