Transcript New Research Points to Prevalence of Acinetobacter

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
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3
3
Researchers’/Presenters’
Names
1School of Health and Bioscience, University of East London, UK, 2 Dept 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
The antimicrobial activity of a novel aqueous allicin extract (AB1000) was tested against 11
DRABs isolated from patients at the Royal London Hospital. Standard antibiograms were
carried out in the clinical laboratories.
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1.00E+06
Aqueous extract of Allicin was prepared 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 Mueller-Hinton (all Oxoid
Ltd,UK). Individual plates were lawned with each test organism using the British Society for
Antimicrobial Chemotherapy (BSAC) standard methods. 6mm holes were then cut in the centre
of each plate and 100ul of each dilution of allicin liquid was added to the hole. Plates were
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).
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 1000 mg l-1 were made
up in aqueous solution. Zone diameters on Oxoid Mueller 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 62.5
mg 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.
cfu/ml
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
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1.5
0.5
0
10
0
Time(hrs)
15
5
0
10
5
15
15
20
25
LH3
LH4
LH6
Discussion and Conclusions
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2
1
50
Abs
Abs
2
10
Figure 1 shows a clear Allicin peak that elutes at 9.8 minutes. Figure 2 shows that in
liquid culture, growth is inhibited at 62.5mg l-1 but below that some strains grow more
slowly at 31 or 15 mg l-1. Using 500 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 iso-sensitest
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. There was no relationship between the antibiogram of the
DRAB and allicin activity
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 is an important nosocomial pathogen. It is ubiquitous in the environment
able to grow at wide range of temperatures and pHs. It is commonly found in soil and
water and also on the skin and distal urethra of healthy people. Acinetobacter has a very
high level of resistance to antimicrobials, and relatively few antibiotics are active against it.
It was such a strain that caused problems with wound infections in injured troops in Iraq 1.
The respiratory tract is the most common site of infection. Acinetobacter can cause a
nosocomial pneumonia, often associated with ventilator use. Other predisposing factors for
Acinetobacter pneumonia include staying in intensive care especially when factors such as
surgery, intubation, tracheostomy, underlying pulmonary disease and antibiotic therapy are
involved. Allicin is the main biologically active agent produced in garlic. It is normally very
unstable however our aqueous extract is not e.g. stable for up to 2yrs as a powder . We
have previously demonstrated that it is active against multiply drug resistant bacteria such
as MRSA 2, 3 and this study is the first report of its activity against DRAB
Figure 3: Comparative Killing curves
against 4 strains of DRAB
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15
This is the first report that aqueous allicin extract is both bacteriostatic and bacteriocidal against
DRAB. 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 of 31 to 62mg
l-1. Although DRAB infections are normally rare, they are an increasing problem and, when they
do occur, are very difficult to treat. Similar to Pseudomonas, DRAB can cause serious
postoperative pneumonia and is able to form biofilms in the body making them even more
difficult to treat. In relation to this capability and the potential effect on therapy and as garlic
extracts have been identified as quorum sensing inhibitors, we are further investigating the
effect of aqueous allicin extract alone and in combination with standard antibiotics on growth of
DRAB and on its formation of biofilms.
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 DRAB strain LH3 (left) and LH6 (right)
showing the effects on growth of different concentrations of allicin
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